Plasma Membrane Compartments and Cytokine Signaling

The cell (plasma) membrane is a complex array of lipids and proteins that plays an essential role in the communication between the cell and its external environment (see image below).  In complex multi-cellular organisms, individual tissue cells communicate with vast varieties of factors including hormones and cytokines that are found in the tissue fluids that bathe cells and within the circulatory system that delivers nutrients to these cells.

Cytokines are a special class of compounds including interferon (IFN) (see structure below) and interleukins.  These substances are low molecular weight proteins often bound to sugar molecules (glycoproteins) that promote cell to cell communication within the immune system in response to inflammation, infectious agents and trauma.

Interferon

Changiiang You and his colleagues from *the Universitat Osnabruck, Germany, studied in detail the interaction dynamics of the complex array of signaling components and receptors on the plasma membrane especially in regard to IFN.  Interferons augment the immune system response to viral infections and impede the growth of cancerous cells through the regulation of specific genes whose gene products impact cell growth.

The results of their extensive investigation demonstrated that microcompartmentalization plays an essential role in facilitating binding of IFN to its specific receptor on the plasma membrane and the resulting cascade of signaling events that leads to gene expression (see image below).  They found that the maintenance of the integrity of the membrane skeleton (MSK) is of crucial importance.

These finding are of value; because, they help elucidate the complex interactions that are required to insure the successful role of key factors like interferon in proper cell function.

Site Specific Phosphorylation of Tau Inhibits Alzheimer’s in Mice

Alzheimer’s disease impacts many individuals in the latter stages of life.  It is a form of dementia that is very devastating to the individuals affected and their families.  On the molecular level, the disease is indicated by extracellular protein “plaques” that are composed of amyloid-β (Aβ) and an accumulation of so-called, “neurofibrillary tangles” within the neurons (see images below).  These tangles are composed of tau protein.  Amyloids represent aggregates of proteins that are stuck together and pose problems for cellular health and development in tissues.  This is especially important within the central nervous system since communication between neurons is of such vital importance.

The accumulated evidence suggests that it is the combined impact of Aβ and tau that lead to the neuronal dysfunction that is indicated by the severe cognitive deficits exhibited by individual with this disease.  Furthermore, the production of Aβ seems to trigger the subsequent phosphorylation of tau protein that lead to its deposition and ultimately to cognitive deficits.  In mouse animal models, the depletion of tau prevents Aβ toxicity.  It seems that by the time the symptoms are evident, significant cell damage has already occurred.

However, recent studies by Dr. Arne Ittner and his colleagues from the School of Medical Sciences at UNSW Australia have shown that site-specific phosphorylation of tau directed by the neuronal p38 mitogen-activated protein kinase enzyme (p38γ) actually inhibited Aβ toxicity.  Furthermore, the depletion of p38γ worsened cognitive defects and increasing p38γ eliminated these deficits.

 

These results are of importance; for, they demonstrate that phosphorylation of tau may or may not be detrimental depending upon the site(s) on the protein structure that are impacted.

The Impact of Climate Change on Desertification

Climate Change and Desertification - The following is a link to an extensive New York Times article regarding the expansion of the Tengger desert in China and the impact of this change on the affected population - 

Integration of Newly-Formed Neurons within the Adult Human Brain

The learning process that proceeds within the adult human brain has long been an intense interest of study for neuroscientists especially in regards to the events that unfold on a cellular and molecular level.  It has been shown that physical activity as well as the experience that comes with being in novel environments triggers the production, development and eventual connectivity of newly-formed neurons within the adult human brain.

Diego D. Alvarez and his colleagues (Laboratorio de Plasticidad Neuronal, Fundación Instituto Leloir–Instituto de Investigaciones Bioquímicas de Buenos Aires–Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina) focused their studies on the mechanism through which the experience of an enriched environment (EE) impacts the incorporation of newly-formed adult neurons into the hippocampal network – an area of the human brain involved in retaining declarative (explicit) memory.  Declarative memory refers to the memory of facts and events.

The cells of interest are referred to as granule cells (GCs) (see image below).  In the investigators’ study of newly-formed GCs using the mouse animal model, they found that exposing the test animal subjects to EE accelerated the incorporation of these neurons into the microcircuits of the dentate gyrus – the apparent site for the establishment of new memories.

Granule Cells

In addition, further study demonstrated that in order for this incorporation of new GCs to proceed, the process required the participation of parvalbumin y-aminobutyric acid-releasing interneurons (PV-INs).  Inactivation of PV-INs effectively prevented the effects of EE.  Neuronal stem cells (NSC) of the adult hippocampus are the precursors of GCs of the dentate gyrus.  

  

These results further our understanding of the complex cellular and molecular mechanisms involved in incorporating new memories in the adult human brain.

Natural Killer Cells in the Treatment of Cancer

It has been well established that the human immune system has the inherent capability to recognize and eliminate aberrant tissue cells in the body that have become transformed into cancerous cells capable of metastasis.  The natural process of finding and eliminating these cells is referred to as cancer surveillance.  It is this understanding that has led to the development of clinical approaches taking advantage of this phenomenon.  The techniques employed in this regard are classified under the heading, cancer immunotherapy.

There is a particular subset of circulating immuno-competent white blood cells referred to as natural killer cells that play a significant role in the body’s response to infectious pathogens as well as in cancer surveillance (see image below).  Dr. Rizwan Rowee and is colleagues from the Department of Medicine, Oncology Division Washington University School of Medicine in St. Louis investigated the properties of natural killer cells in regard to their ability to identify and destroy cancerous cells.  They were particularly interested in their “memory” capacity.  It has long been known that subsets of immuno-competent cells retain the capacity to recognize and attack particular targets from previous encounters.  This is an indispensable feature of a normal immune system.

Natural Killer Cells Attacking a Target Cell

Following exhaustive study and analysis of these natural killer cells with human leukemic cells in culture, the investigators discovered that natural killer cells with memory-like capability actually demonstrated an anti-leukemic effect against patients with acute myeloid leukemia (AML).  In some cases, this treatment led to clinical remissions.  The investigators reported that, “Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.”

These are very important findings especially in regard to the treatment of patients with AML; since, this disease is a particularly aggressive form of leukemia.

Dr. Arnoldo Gabaldón

Malaria is one of the world’s deadliest diseases.  It is especially prevalent in the tropics.  The life cycle of the causative microbial parasite – members of the plasmodium genus i.e. Plasmodium vivax is complex involving the Anopheles mosquito as a vector (see images below).  The nature of the infection is such that it has eluded the development of an effective vaccine for many years. 

Human red cells infected by Plasmodium vivax

In light of this, it is quite surprising that Dr. Arnoldo Gabaldón, born in 1909, in Venezuela made a significant contribution to the understanding of this disease and its implications in regard to public health.

Gabaldón earned a doctorate in medical sciences at the Universidad Central de Venezuela. He continued his education internationally working in Hamburg and the United States at  the Rockefeller Foundation and ultimately received a doctorate from Johns Hopkins University in hygiene sciences with a specialty in protozoology. With this kind of medical background and expertise in infectious disease, he was asked to head the newly created Special Directorate of Malariology in his home country of Venezuela in 1936.  He held this post until 1950.

He successfully applied his understanding of the methodologies required to combat infectious disease to the rate and severity of malarial infection that gripped his country in the 1930s.  This included the emphasis on public hygiene and sanitation and the judicious application of anti-malarial drugs. His approach was so successful that mortality resulting from malarial infection was decreased significantly by 1944 and, more importantly, its control was seen as within reach.

This initial success was followed by an attempt to significantly reduce the Anopheles mosquito population.  For this purpose, the insecticide dichlorodiphenyltrichloroethane  (DDT) was used.  The historic data has revealed that “by 1950 the death rate from malaria in the country had been reduced to 9 per 100,000 inhabitants and was eradicated in an area of 132 000 km2. In 1955, 10 years after the program started the rate was lowered to 1 per 100 000 population and the eradicated area had increased to 305,414 km2.” On balance, it should be kept in mind that the discovery of the ecological burden posed by the use of DDT on the natural environment has effectively banned its application for many years.

Gabaldón is also credited with discovering a new species of malarial parasites and had focused a great deal of his efforts on further study of the Anopheles mosquito.  He was later appointed Minister of Health and Welfare between 1959 and 1964 in recognition of his premier understanding regarding issued of public health.  He died in September of 1990.

Arnoldo Gabaldón made a significant contribution to the principles and practices of public health around the area of infectious disease.  The example of his leadership has been emulated throughout the world and possibly has saved countless lives. 

The C9ORF72 Protein as a Suppressor of Autoimmunity

There are many chronic illnesses that are referred to as autoimmune diseases such as multiple sclerosis, amyotrophic lateral sclerosis (ALS) and pemphigus vulgaris for example; the etiology of these diseases originates from the patient’s immune system attacking normal tissue.   

ALS is a disease that presents as a progressive degeneration of the normal function of motor neurons resulting in paralysis and death.  It has been shown that mutations in the C9ORF72 gene are commonly found in ALS and frontotemporal dementia.  This gene is located on the short arm of chromosome 9 in humans.  This gene contains the information for the production of the C9orf72 protein.  Furthermore, in animal model studies using the mouse, it has been found that mutations in this gene result in Autoimmune-like symptoms.  Transplantation of normal murine bone marrow positively impacted diseased mice with this mutation and bone marrow transplantation of diseased mice into normal animals alternatively resulted in symptoms of autoimmunity.  These latter results led investigators to suspect that the C9orf72 gene is normally active in hematopoietic cells – cells residing in the bone marrow that produce circulating blood cells - in suppressing autoimmunity.

Recent findings have clearly established that mice that harbor mutations in the C9orf72 gene that result in the loss of function of the C9orf72 protein gene product develop the following symptoms –

• splenomegaly – enlarged spleen
• neutrophilia – increased population of neutrophils in the circulation
• thrombocytopenia – decreased numbers of platelets
• increased expression of inflammatory cytokines
• severe autoimmunity.

These diseased animal suffer a high mortality rate.

These data represent strong evidence that the C9ORF72 gene plays a very important role in the suppression of autoimmunity and may prove to be of future therapeutic value.

What Will be Our Legacy?

The essential questions I would like to pose are the following-

• Are we prepared to leave as our legacy not only to future generations of humans, but also the future of our fellow creatures a world whose living populations will be severely decimated by the onslaught of climate change brought on by human activity?
• Are we prepared to jeopardize the quality of life and security of future generations of humanity on planet Earth so that we can maintain our privileged and comfortable lifestyles based upon a patent disregard for the environmental degradation that we necessarily impose upon our planet?

These questions are no longer simply philosophical in nature.  The current data is clear that we are in the midst of a major change in the global environment caused primarily by human activity.  The following statement was made within the Smithsonian Museums website – Smithsonian.com – “According to the International Union of Geological Sciences (IUGS), the professional organization in charge of defining Earth’s time scale, we are officially in the Holocene (“entirely recent”) epoch, which began 11,700 years ago after the last major ice age.

“But that label is outdated, some experts say. They argue for “Anthropocene”—from anthropo, for “man,” and cene, for “new”—because human-kind has caused mass extinctions of plant and animal species, polluted the oceans and altered the atmosphere, among other lasting impacts.”

Humanity’s response, within the 21^st century, to this real crisis will determine the future that awaits the living world.  Of course, the earth has been through at least five major mass extinctions in its long geological history and will certainly survive another.  The kind of adjustments involved, however, require long stretches of time involving many thousands if not millions of years.  We do not have the luxury of waiting.  The future of the natural environment depends upon what we do, or what we fail to do now.

The Role of the Enzyme Acid Ceramidase in the Etiology of Cancer

Ceramide belongs to a class of bio-active organic compounds referred to as sphingolipids.

  

This class of compounds has been shown to play important roles in many cellular processes including cell growth and proliferation, growth arrest and most importantly, apoptosis – programmed cell death.  Furthermore, research studies have strongly implicated sphingolipid signaling dysfunction in tumor progression – metastasis.  In addition, sphingolipids seem to play a role in the effect of chemotherapy and radiation upon cancer progression in patients with metastatic disease.

The current data strongly suggests that the enzyme acid ceramidase (AC) is implicated in both tumor growth and resistance to various therapeutic modalities as suggested above.  AC is an enzyme that catalyzes the breakdown of ceramide to sphingosine and fatty acid (see diagram below).  

AC over expression has been found in association with certain metastatic cancers including prostate cancer.  A decrease in the intracellular levels of ceramide has been shown to result in a decrease in apoptosis that would ordinarily lead to cell death for proliferating tumor cells.

As a consequence of these data, Daniele Piomelli, the Louise Turner Arnold Chair in Neurosciences at UCI, and colleagues at the Italian Institute of Technology, have also established an association with over expression of AC and metastatic disease in melanoma patients.  This group has proceeded to successfully develop drugs that effectively inhibit AC in an attempt to restore an appropriate level of apoptosis and slow down tumor progression.

This approach is experimentally sound and has been shown to be efficacious in in-vitro experiments with cancerous cells in culture.  This 

Last Universal Common Ancestor

It has long been suspected that the progenitor of life on planet earth most likely came from the sea.  It has been proposed that the possible origins of life could have been in shallow pools or under more extreme conditions such as within deep-sea vents or proximal to active volcanoes.

The current domains of life consist of bacteria –prokaryotes, the archaea -found in deep sea vents and the eukaryotes that comprise all the animal and plant life on the planet.  It has also been proposed that the archaea and the bacteria preceded the more complex eukaryotic cell type.

William F. Martin, an evolutionary biologist, from Heinrich Heine University in Düsseldorf, Germany, focused his research efforts on finding the progenitor of archaea and bacteria.  To do this, the known genetic structure of members of the archaea and bacteria domains were extensively examined.  This involved the examination of some six million genes representing thousands of microbes.

From these data, Martin and his colleagues were able to construct evolutionary family trees and were able to deduce that 355 gene families originated from single cell bacteria-like organism.  That organism is referred to as the Last Universal Common Ancestor (LUCA).  Furthermore, it is believed that LUCA lived some four billion years ago when the young earth was barely 500 million years old.

If this conclusion is correct, it clearly proposes that life began very early in the evolution of the planet earth and that the evolution of life was a much longer process than previously envisioned.

Myelodysplastic Syndromes (MDS)

Myelodysplastic syndromes (MDS) represent a disease state that has its origin in the bone marrow.  Within the bone marrow are resident stem cells that are the progenitors of the circulating white and red cells and platelets that play vital roles in immune function (white cells), oxygenation of the tissues (red cells) and the ability of blood to clot as the result of trauma (platelets) - see diagram below.  The wide range of types of human leukemia are cancers of these progenitor stem cells.

In the case of MDS, these stems fail to mature and produce cells that are referred to as blasts and so-called dysplastic cells.  As a result, the number of mature stem cells are diminished in number; a state that results in diminished numbers of circulating white (neutropenia) and red cells (anemia) and platelets (thrombocytopenia).  It is this diminished cell count that produces the symptoms associated with MDS.  These symptoms may include –

·         Fatigue

·         Weakness

·         Easy bruising or bleeding

·         Fever

·         Bone pain

·         Shortness of breath

·         Frequent infections.

There are also cases in which the overall cell counts appear normal, but the circulating cells are abnormal.  Chromosomal abnormalities have also been found associated with MDS.  What makes MDS particularly significant from a human health perspective, is that certain subtypes of MDS have been shown to be a precursor to acute myeloid leukemia (AML) – a particularly aggressive form of leukemia

From the perspective of scientific research, the question that remains to be answered is what are the cause(s) of MDS.  This question has been and continues to be the focus of a concerted and intense collaborative effort.

A Very Promising Treatment for Patients with Glioblastoma

Glioblastoma is an exceedingly aggressive cancer of the brain that usually leads to death within months of its initial diagnosis. However, in clinical trials conducted by Drs Gordana Vlahovic and Matthias Gromeier at the Preston Robert Tisch Brain Tumor Center at Duke University in Durham, NC, amazing results have been achieved in patients with this disease using a human-engineered polio virus that has been stripped of its ability to cause disease. This modified virus with the ability to kill cancer cells (oncolytic) is referred to as PVS-RIPO (see illustration below). The results to-date have been so impressive, that the Federal Drug Administration (FDA) has accelerated the approval process for its use in clinical settings opening up its application to perhaps hundreds of patients who would otherwise face a grim future.

PVS-RIPO

PVS-RIPO was engineered by removing the genes that are responsible for causing the neuronal damage that produces the deleterious effects of polio in humans and added in its place a portion of the genetic code of the ubiquitous cold-causing rhinovirus.


The rationale for this approach is multi-faceted. The polio virus has a natural receptor that can attach to many different forms of cancer. This receptor binds to CD155. CD155 is a cell surface glycoprotein that spans the outer cell membrane (transmembrane). It is often referred to as the Poliovirus Receptor (PVR). It has been established that CD155 plays a role in the establishment of intercellular junctions between epithelial cells. It seems that CD155 is over-expressed and abundant in various kinds of cancers including glioblastoma and pancreatic cancer.  The strategy employed was to use PVS-RIPO to preferentially infect the patient's cancerous glioblastoma cells.


In the case of the patients involved in clinical trials suffering advanced stages of glioblastoma as mentioned above, PVS-RIPO was infused directly into the tumor in order to maximize exposure to the virus. It seems that the amazing success of this treatment is due not only to successful infection with PVS-RIPO but also the subsequent mobilization of the immune system against the cancer cells.

This approach to treating advanced glioblastoma may ultimately extend to the treatment of other intractable cancers such as pancreatic cancer. This is an exceedingly exciting development that may have broad application in the treatment of and ultimate cure of cancer.

Origin of Life on Planet Earth

A question that has plagued scientists for a long time is centered around the question as to how life began on planet earth.  It is well known that life is found even under the most inhospitable conditions – a stunning example of this is that life exists within the deep sea hydrothermal vents that are found on the ocean floor.  In addition, microorganisms such as Hormoconis resinae contaminate jet fuel – using this hydrocarbon source as a vital nutrient – and are known to cause corrosion in the tanks that hold this fuel.

Therefore, it can be reasonably postulated that simple life forms could thrive in the harsh conditions of prebiotic earth when oxygen was not present within the atmosphere at that time.  But the question remains as to how did life begin – what was the process by which self-sustaining living organisms were formed from rudimentary compounds.

It has been shown that in an artificial environment created in the laboratory  in which an atmospheric environment was created to simulate the conditions believed to have existed in the prebiotic world, the addition of an energy source – such as lightening – produced rudimentary organic compounds found in living cells.  These experiments were conducted in the 1950's, by the biochemists Stanley Miller and Harold Urey.  These results are only suggestive evidence that organic compounds could have been created spontaneously in the early-earth environment.  It is, of course, far from the complete story.

Since DNA and RNA are fundamental ingredients to all of life as we know it and capable of self-replication, a key step in the evolution of life would be the conversion of simple organic compounds to purines that are some of the important building blocks for both DNA and RNA and for the synthesis of Adenosine Triphosphate (ATP) – the molecule that is responsible for trapping energy derived from metabolism for all of life.

Furthermore, the preponderance of evidence now suggests that RNA may have preceded DNA as the repository of genetic information capable of self-replication.  Certain forms of RNA also demonstrate catalytic properties (ribozymes) that are essential to sustain life.  Of course, contemporary advanced cell structure uses a host of enzymes to accomplish essential catalytic functions.

 

RNA is made of four different nucleobases  -two pyrimidines – cytosine and uracil – and two purines adenine and guanine.  Previous work done by John D. Sutherland from the School of Chemistry, University of Manchester, UK has shown a plausible synthetic route to pyrimidines in an abiotic environment.  But the route to purines has been more elusive.

Recent work by T. Carell from the Department of Chemistry, Ludwig-Maximilians University Munich, Germany and fellow investigators has recently demonstrated a mechanism that could account for spontaneous creation of purines from simpler compounds readily available within the natural environment of early earth.  The pathway involves the spontaneous synthesis of aminopyrimidines from hydrogen, cyanide and water – compounds readily available in the early earth environment.  Although aminopyrimidines can produce a wide range of synthetic products, in an environment of formic acid, the predominant product is formamidopyrimidine (FaPy) known to readily produce purines.  Furthermore, formic acid has been shown to be present in comets that collided frequently with earth during the early stages of its evolution.

Formamidopyrimidine

Although this work is very significant, it does not explain how purines and pyrimidines would lead to the creation of more complex and sophisticated RNA molecules.  Nor does it shed any real light on the requisite formation of a cellular environment for biosynthetic reactions so necessary for the containment and sustenance of life processes.

Successful Treatment of B-Cell ALL Using Adoptive Cell Transfer

The standard treatment of cancer patients has consisted of an approach involving some combination of surgery, chemotherapy and radiation.  Admittedly, these methodologies have grown sophisticated over the years especially in the areas of surgery and radiation.  However, chemotherapy is a “shotgun approach” employing powerful drugs that target any dividing cells.  The nature of these drugs cause significant side-effects in the patients that they are administered to.

There are a new family of drugs that have been developed to combat certain types of cancers that are more highly targeted.  Among these are Imatinib Mesylate – Gleevic –(see illustration below).  Gleevic has been used in the treatment of acute lymphoblastic leukemia (ALL) and gastrointestinal stromal tumor.  This drug specifically targets the enzyme tyrosine kinase that has been implicated in certain cancers. 

 Gleevic

Another drug that has shown promise in trastuzmab – Herceptin (see illustration below).  Herceptin has been used to treat patients with HER2+ breast cancer and adenocarcinoma of the stomach, for example.  Herceptin is a monoclonal antibody (mAb) that targets the HER2 protein found on certain types of cancer cells.

Herceptin

The new approach to fighting certain types of cancers involves a methodology referred to as adoptive cell transfer (ACT).  ACT actually employs the patient’s own immune system in fighting the cancer cells.  Impressive results using this methodology has been shown in clinical trials involving patients with advanced B-cell ALL.

The cancerous B-cells in ALL have a protein on their cell surface that is referred to as CD19.  This protein makes an appropriate target for immunotherapy.  The rationale behind ACT is to utilize the patient’s own T-cells to selectively kill ALL B-cells bearing this marker.  The following steps have been successfully employed in this treatment –

• CD4 and CD8 lymphocytes are harvested from the patient’s blood.  CD4 T-cells are so-called helper cells and the CD8 T-cells are the cytotoxic cells.
• These cells are genetically modified using an engineered retrovirus.  The genetic information that is introduced results in the production of a protein referred to as a chimeric antigen receptor (CAR) that binds to a specific cancer cell surface protein (see illustration below).  Structurally, CARS are modified mAbs.  In the case of ALL, the target protein is CD19.
• Once the patient’s CD4 and CD8 lymphocytes have been successfully modified in the laboratory, they are grown out into billions of cells.  These are the cells that are reintroduced into the patient via infusion.  

CAR Signaling

There is one significant side effect from this approach; it is referred to as cytokine release syndrome that is a direct result of the modified T cells actively involved in killing their cancerous targets.  However, this is a manageable condition.

The results to date in clinical trials have been extraordinary.  This methodology shows great promise that eventually may have broader applications in the treatment of cancer.

New Danger Posed by the Increasing Abuse of the Opioid Drug - Fentanyl

Opioids are a class of compounds that relieve pain by binding to opioid receptors found on neurons that send signals to the brain that the brain interprets as pain; this binding results in a significant reduction in these signals.  In addition, opioids also bind to analogous receptors within the brain that reduces the emotional response to painful stimuli.

 

According to the National Institute of Drug Abuse, “Medications that fall within this class (opioids) include hydrocodone (e.g., Vicodin), oxycodone (e.g., OxyContin, Percocet), morphine (e.g., Kadian, Avinza), codeine, and related drugs. Hydrocodone products are the most commonly prescribed for a variety of painful conditions, including dental and injury-related pain. Morphine is often used before and after surgical procedures to alleviate severe pain. Codeine, on the other hand, is often prescribed for mild pain. In addition to their pain-relieving properties, some of these drugs—codeine and diphenoxylate (Lomotil) for example—can be used to relieve coughs and severe diarrhea.”

These receptors pre-exist in nature for they bind to certain endogenous opioids such as dynorphins, enkephalins and endorphins.  The structures of morphine and an endorphin are shown below

Morphine

Endorphin

Opioids are readily found in nature and, as such, have been used to relieve for thousands of years within human populations.  Biochemically, opioids receptors (see illustration below) that are imbedded in the outer cell membranes of target cells are G-protein coupled, and activate inhibitory G-proteins. Once bound to the appropriate receptor, they trigger a series of cascading chemical events within the cell resulting in the suppression of neuronal signaling.

Opioid Receptor

Although opioids play a very important role in modern medicine for the relief of acute pain, there is an alarming rise of abuse of these substances especially since they are highly addictive.  Of special concern is the increased street-use of a very potent opioid – fentanyl (see structure below)

Fentanyl

Fentanyl is, often used in anesthesia to prevent pain after surgery or other procedures.  According to DEA administrator Miechele M. Leonhard "Drug incidents and overdoses related to fentanyl are occurring at an alarming rate throughout the United States and represent a significant threat to public health and safety."

"Often laced in heroin, fentanyl and fentanyl analogues produced in illicit, clandestine labs are up to 100 times more powerful than morphine and 30 to 50 times more powerful than heroin," she added.  In addition, the DEA has noted that, “ingestion of even small doses ― as small as 0.25 mg ― can be fatal. Its euphoric effects are indistinguishable from those of morphine or heroin.”

Of additional concern is the fact that fentanyl can be absorbed directly through the skin, or unknowingly inhaled as an airborne powder; this is especially of critical importance for law enforcement personnel.  Another cause for concern is that fentanyl - like other compounds in the opioid class - can be readily synthesized. In an appropriately equipped laboratory.

The abuse of opioid substances, especially fentanyl is a very real, immediate and important public health issue and deserves the attention of not only government agencies but concerned citizens.  Rather than focusing on criminal punishment of offenders, greater emphasis and resources should be brought to bear on drug rehabilitation and education as a way to help prevent such abuse from happening in the first place.

Creation of a Synthetic Organism with the Smallest Complement of Genes

The tools available to molecular biologists especially in regard to gene sequencing and assembly allow investigators to produce nucleotide sequences that incorporate specific genes and gene clusters into DNA created in-situ.  A team of investigators headed by Craig Venter from the J. Craig Venter Institute in La Jolla California in effect created a microorganism containing 473 genes (Syn 3.0).  The purpose of this investigation was to determine the minimal amount of genetic material required to sustain life as an autonomous organism and successfully reproduce. 

In 2010, Venter and his colleagues created an entire chromosome from the bacterium, Mycoplasma mycoides (this organism has only one chromosome) and demonstrated that this synthetic chromosome was completely functional.  They did this by stripping out the naturally occurring DNA from the mycoplasma, M. capricolum and replacing it with the synthetic chromosome.  The modified organism was called Syn 1.0 and with its complement of 901 genes was shown to be completely viable and capable of reproduction.

With this material in hand, the investigative group sought to assemble Syn 3.0 by methodically whittling down the DNA in Syn 1.0 to the smallest number of genes required to sustain life. The result of this painstaking work was Syn 3.0

What makes this current result so remarkable is that this organism is entirely new.  Of the 473 genes, 149 (31.5%) are of unknown function; therefore, additional work will focus on the discovery of the function of these apparently essential genes.  Syn 3.0 may prove to be an invaluable tool in understanding the evolution of life on planet earth.

What is the A1C Test?

The disease diabetes mellitus occurs in two different forms – juvenile or adult-onset.  In either case, the source of the illness is lack of or reduced production of the hormone insulin (see image below) whose role is to enhance the uptake of glucose circulating in the blood by tissue cells, especially adipose and skeletal muscle.  Insulin (as seen below) is referred to as a globular protein

Insulin is normally produced by specialized beta cells resident in the Islets of Langerhans within the pancreas.  It has been established that juvenile diabetes is an auto-immune disease in which the immune system of the patient attacks these beta cells.  Adult-onset diabetes, on the other hand, has a strong association with obesity.

This inability to transfer glucose to tissue cells where it is utilized for energy, leads to high concentrations of glucose in the blood (hyperglycemia).  Over a prolonged period of time, this hyperglycemic state results in very serious and ultimately life-threatening complications including blindness, impaired kidney function, cardiovascular issues leading to heart trouble and leaving victims prone to amputation.  These deleterious side effects arise as the excess glucose in the blood reacts with proteins in various tissues throughout the body – this biochemical reaction is referred to as glycosylation.

Once the cause of diabetes was discovered, it was realized that an obvious therapeutic approach is to give the patient insulin from an external source.  Before the advent of DNA recombinant technology, patients were given insulin harvested from cow pancreas (bovine insulin).  Bovine and human insulin are close enough in structure to allow bovine insulin to have an efficacious effect.  Currently, of course, human insulin is readily available.

Over many years of treating diabetic patients with human insulin, it was realized that periodic administration of insulin is not comparable to the body’s finely tuned regulation of insulin production so as to maintain optimal levels of blood glucose.  In response to this deficit, a technology arose to employ the use of an insulin pump in order to maintain a steady stream of insulin along with careful monitoring of blood glucose levels.  In addition, considerable emphasis has been placed on nutrition, exercise and weight control especially in regard to adult-onset diabetes.

Another important tool in the treatment of diabetes is the so-called A1C test.  This test provides information as to the average level of blood glucose over a 3-month time span; it is also referred to as the glycohemoglobin test.  This test measures the amount of glycosylated hemoglobin.  Hemoglobin is the specialized protein in red blood cells that is designed to carry oxygen to the tissues.  This protein reacts with excess glucose in the blood.  Since red blood cells are recycled by the body over a 3-month period, the test provides information about the average level of glucose in the blood over this period of time.  The result of this test is reported as a percentage.  The normal value is about 5.7%.  The higher the percentage, the higher the blood glucose level has been over the past 3 months.

The A1C test is an important tool in determining the efficacy of treatment for diabetic patients.  

The Role of the Large Protein Titin in Dilated Cardiomyopathy

There is a serious heart condition referred to as dilated cardiomyopathy (DCM) that is a major cause of heart failure and often results in premature death; this disease is found in one in two-hundred and fifty adults (0.4%).  DCM can originate either as a result of an underlying vascular problem or can have a genetic origin.  This report will focus on the progress that has been made in regards to the genetic implications of this condition.

Through the work of Dr. John T. Hinson at the Division of Cardiovascular Medicine at the Brigham and Women’s Hospital, Boston MA and his colleagues from many diverse institutions, it has been show that mutations of a large protein that constitutes one-half of the sarcomere (a structural unit of a myofibril in striated muscle) are the most common cause for DCM.  This protein is referred to as titin (TTN) (see image below) and the mutations involved result in a truncated version of TTN.  These genetic variants of TTN are referred to as TTN-truncating variants (TTNtvs).

Since the functional significance of TTN within the muscle sarcomere was unclear, the investigators involved in this research, applied the tools of molecular biology to better define the role played by TTN within heart muscle.  To accomplish this, they grew out cardiac micro-tissue cultures comprised of cardiomyocytes derived from pluripotent stem cells (iPS) that were harvested from the patients studied.  When these patient-derived cells were compared to those derived from normal individuals (the controls), it was discovered that, “certain missense mutations like TTNtvs diminish contractile performance and are pathogenic.”

Furthermore, these patient-derived cardiomyocytes also demonstrated sarcomere insufficiency, reduced responses to mechanical and biochemical stress as well as impairment in critical cell signaling pathways.  All of these results, when taken together, point to TTNtvs as playing a causative role in genetically-induced DCM.  This is a significant finding with broad implications.

Molecular Disease

There are a host of diseases that are a direct result of a mutation in a single gene. Examples of this kind of disease are many including sickle cell anemia, severe combined immunodeficiency disease (SCID) and many others. The world famous chemist, Linus Pauling (1901-1994) coined the term Molecular Disease to refer to this type of illness. He was awarded the Nobel Prize in Chemistry for his work in 1954.

 We shall use sickle cell anemia as a case in point to illustrate how a singular change in the molecular structure of a gene can have profound consequences for the organism. A patient with sickle cell anemia presents the following symptoms often beginning at 4 months old:

• Painful episodes that can last hours or days Attacks of abdominal pain 
• Bone pain 
• Breathlessness 
• Delayed growth and puberty 
• Fatigue 
• Fever 
• Jaundice 
• Paleness 
• Rapid heart rate 
• Ulcers on the lower legs (in adolescents and adults). 

Other symptoms include:

• Chest pain 
• Excessive thirst 
• Frequent urination 
• Painful and prolonged erection (priapism - occurs in 10 - 40% of men with the disease) 
• Poor eyesight/blindness 
• Strokes 
• Skin ulcers. 

This disease was thought to have a genetic etiology based upon the epidemiological data which showed its prevalence among individuals of African descent (one in twelve African Americans are heterozygous for this trait). Furthermore, these data also pointed to a recessive trait i.e. both alleles have to possess the altered gene for the symptoms to appear.

The disease presents with a singular characteristic – misshapen red blood cells (See Figure 1). This change in morphology from the normal disc-shaped cell to crescent-shaped is a direct result of the altered tertiary structure of the hemoglobin molecule (referred to as Hemoglobin S). Normal hemoglobin has a globular tertiary structure (See Figure 2).

Figure 1 

Figure 2   

Hemoglobin is the protein found in circulating red blood cells (RBC) that is responsible for transporting oxygen through the blood stream and ultimately to all the tissues of the body. This change in the structure of hemoglobin is a direct result of a replacement of one of its constituent amino acids. In this case the sixth amino acid in the protein polypeptide chain has been changed from glutamic acid to valine. This singular modification causes the deoxygenated form of the protein to clump together (See Figure 3). This replacement results in a molecule that is no longer readily soluble in the cell cytoplasm of RBC. As a result of this one change, the overall morphology of RBC is changed from the normal disc shape to crescent shape and they can no longer flow readily through the bloodstream as shown in Figure 1.

 Figure 3   

Since the sequence of all proteins is determined by the specific sequence of nucleotides in the genes responsible for their production, sickle cell anemia is a direct result of a single point mutation in the gene carrying the information for the production of hemoglobin found on human chromosome number 16 – hence the term, molecular disease.

 Since this disease has such a serious impact on the mortality for those who are afflicted, it might be asked why natural selection has not deleted this deleterious gene over the course of evolution. There is an explanation for this seeming contradiction. Those individuals that are heterozygous for this trait – where only one of the alleles has the mutation – have a subset of RBC that is misshapen. The remaining cells are normal, and these individuals are symptom free. Those who are homozygous for the trait – where both alleles have the mutation – have the full-blown disease. It appears that those who are heterozygous for the trait are protected from the malaria parasite whose host is RBC. Therefore, natural selection would favor those who are heterozygous for sickle cell anemia (carriers). 

In the past, this type of illness has been impervious to the possibility of a cure, since its origin resides in the very makeup of an individual’s heredity as expressed through the genes. However, since, red blood cells originate in the bone marrow, sickle cell anemia can be cured by bone marrow transplantation, but this approach has its own set of significant risks.

Recent advances in molecular biology and gene therapy have demonstrated that this daunting limitation can be effectively breached using gene therapy. SCID is a particularly devastating and ultimately fatal disease in which the affected child has no defense against infections. Through the ground breaking work of Dr. Alessandro Aiuti, ten patients suffering from SCID are still alive. The mutated gene in this condition is the ADA gene responsible for the production of the enzyme adenosine deaminase - ADA. The laboratory of Dr. Aiuti from the San Raffaele Institute for Gene Therapy in Milan successfully used the following procedure: bone marrow cells from the patients involved were incubated with a specially engineered virus containing the normal ADA gene. These engineered cells were reintroduced into the patients. Positive results were seen almost immediately following treatment. A similar approach has been used in the treatment of a disease characterized by a congenital degeneration of the retina. In this study four of six patients had a notable improvement of vision.

The latest advance has been made with Adrenoleukodystophy (ALD), a disease linked to the X chromosome. This is a severe neurodegenerative disease that leads to destruction of myelin, the outer membrane of nerve cells, in the brain and severe nervous system dysfunction. This disease is caused by a mutation in the ABCD1 gene. The first successful clinical test using gene therapy for ALD has recently been reported by Dr. Nathalie Cartier and her colleagues from the University of Paris-Descartes in Paris, France. The approach used was to take hematopoietic stem cells (HCS) from two young male patients and incubate their cells with a virus that was engineered to carry the normal ABCD1 gene. These modified cells were then reintroduced to the patients. Eventually, blood cells with the normal gene were found distributed throughout each patient’s body. Within 14 to 16 months post treatment, cerebral demyelination was arrested and neurological and cognitive functions remained stable. The patients’ own cells were used in this procedure; this avoids any need for a donor and obviates any concern of possible rejection. This is an extraordinary result and has profound implications for the future of gene therapy in medicine and may eventually find application in the treatment of sickle cell anemia.

Viruses Shown to Transfer Immunity between Infected Cells

Interferons (IFN) are known to play a critical role in the innate immune response to viral infection.  The molecular mechanism responsible for eliciting this response has been well studied.  It has been shown that IFN production is initiated by signaling pathways activated by molecular sensors in the presence of viral particles including cytosolic DNA sensors.  One of these DNA sensors is Cyclic GMP-AMP synthase (cGAS).  This enzyme, when activated, catalyzes the synthesis of a second messenger referred to as cyclic GMP-AMP (cGAMP).  This messenger subsequently activates transcription factors that “turn on” the genes responsible for the production of IFN (STING).

This cascading sequence of steps occurs within the infected cell.  This activation of IFN may also be spread to nearby cells connected via a gap junction.  However, Dr. A. Bridgeman and his colleagues from the medical Research Council Human Immunology Unit at the Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, suspected that this immunity could also be transferred by the viral vector to whatever additional cells it was infecting.  They proposed that the infecting virus might actually incorporate and transfer the cGAMP second messenger.  This suggestion has some precedence in that the human immunodeficiency virus 1 (HIV-1) has been shown to incorporate host-derived substances.  Given this known behavior, the investigators hypothesized that cGAMP could be picked up by infecting virions, incorporated and subsequently passed on to additional host cells and in that way actually inadvertently spread the immune response.

In order to test this proposal, they used modified and attenuated virions to infect a human embryonic kidney cell line that expressed STING and that were able to induce the production of IFN in response to cGAMP.  When these same viruses were subsequently exposed to a target cell line, they were shown to have induced the expression of IFN suggesting that their working hypothesis was correct.  The investigators painstakingly ruled out other possible variables that might explain this phenomenon.  Even virus-like particles stripped of their RNA genome still induced IFN production in target cells.   Finally, they went on to demonstrate unambiguously that cGAMP was, in fact, packaged into the virus studied.

This is an interesting finding that may have clinical value.  In light of this evidence, the authors suggest that “using viral vectors with cGAMP therefore holds promise for vaccine development.”  

The Genomics of Mental Illness

Mental illness covers a wide range of diseases including schizophrenia (SCZ), autism spectrum disorder (ASD), chronic depression and bipolar disorder (BPD).  The aberrant behavior associated with these disorders has long been ascribed to factors other than that of a genetic or organic origin.  On account of the tremendous strides that have been made in the fields of molecular biology and human genetics, there is a new understanding of the role of human genes in the development of psychiatric diseases.

Daniel H. Geschwind and Jonathan Flint from the Department of Neurology, Psychiatry and Human Genetics at the David Geffen School of Medicine in University of California in Los Angeles have published a review article in the Journal Science (Vol 349, No 625, pp 1489-1493) in which they describe the current scientific understanding of the role genes play in mental illness.  Within the body of this review, they make a number of salient points.

Due to remarkable technological advances, variations at millions of Single Nucleotide Polymorphisms (SNPs) within the human genome can be detected.  Furthermore, with the use of microarrays, genome-wide association studies (GWASs) can be performed that can establish associations between disease states and common genetic alleles.  As a result of such exhaustive studies, it appears that GWASs generally lie within regulatory regions of the genome.  Since regulatory sites usually lie within close proximity to the genes that are regulated, it is not unreasonable to assume that it is such functional genes that are affected.

 

In addition, microarrays have also identified copy number variants (CNVs) associated with both SCZ and ASD.  These CNVs are the result of either a gain or loss of DNA involving DNA segments > 1 kilobase (kb) in size.   Another area of intense investigation involves genomic sequencing that focuses on the complete protein coding sequence also referred to as whole-exome sequencing (WES).  WES reveals the DNA sequences that have the coding information within the entire genome for all the proteins destined for production.  To date, tens of thousands of individuals have been analyzed in this way.  From this extensive data, rare protein variants have been shown to be associated with SCZ and ASD.

Although the application of these methodologies have contributed greatly to the understanding of the role of genes in mental illness, the various mental illness disease states appear to involve a multiplicity of genetic loci making it difficult to pinpoint the precise etiology of the disease process.  However, great progress continues to be made in this area of research, making it more likely that complete molecular mechanisms will eventually be uncovered.