Molecular switch model for cardiomyocyte proliferation.

This review deals with the human adult cardiomyocyte proliferation as a potential source for heart repair after injury. The mechanism to regain the proliferative capacity of adult cardiomyocytes is a challenge. However, recent studies are promising in showing that the 'locked' cell cycle of adult cardiomyocytes could be released through modulation of cell cycle checkpoints. In support of this are the signaling pathways of Notch, Hippo, Wnt, Akt and Jak/Stat that facilitate or inhibit the transition at cell cycle checkpoints. Cyclins and cyclin dependant kinases (CDKs) facilitate this transition which in turn is regulated by inhibitory action of pocket protein e.g. p21, p27 and p57. Transcription factors e.g. E2F, GATA4, TBx20 up regulate Cyclin A, A2, D, E, and CDK4 as promoters of cell cycle and Meis-1 and HIF-1 alpha down regulate cyclin D and E to inhibit the cell cycle. Paracrine factors like Neuregulin-1, IGF-1 and Oncostatin M and Extracellular Matrix proteins like Agrin have been involved in cardiomyocyte proliferation and dedifferentiation processes. A molecular switch model is proposed that transforms the post mitotic cell into an actively dividing cell. This model shows how the cell cycle is regulated through on- and off switch mechanisms through interaction of transcription factors and signaling pathways with proteins of the cell cycle checkpoints. Signals triggered by injury may activate the right combination of the various pathways that can 'switch on' the proliferation signals leading to myocardial regeneration.

Implications of genetic variation of common Drug Metabolizing Enzymes and ABC Transporters among the Pakistani Population.

Genetic polymorphism of drug metabolizing enzymes and transporters may influence drug response. The frequency varies substantially between ethnicities thus having implications on appropriate selection and dosage of various drugs in different populations. The distribution of genetic polymorphisms in healthy Pakistanis has so far not been described. In this study, 155 healthy adults (98 females) were included from all districts of Karachi. DNA was extracted from saliva and genotyped for relevant SNVs in CYP1A1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 as well as ALDH3A1, GSTA1, ABCB1 and ABCC2. About 64% of the participants were born to parents who were unrelated to each other. There was generally a higher prevalence (p < 0.05) of variant alleles of CYP450 1A2, 2B6, 2C19, 3A5, ALDH3A1, GSTM1 as well as ABCB1 and ABCC2 in this study cohort than in other ethnicities reported in the HapMap database. In contrast, the prevalence of variant alleles was lower in GSTA1. Therefore, in the Pakistani population sample from Karachi a significantly different prevalence of variant drug metabolizing enzymes and ABC transporters was observed as compared to other ethnicities, which could have putative clinical consequences on drug efficacy and safety.

Evolution of pH buffers and water homeostasis in eukaryotes: homology between humans and Acanthamoeba proteins.

AIM: This study intended to trace the evolution of acid-base buffers and water homeostasis in eukaryotes. Acanthamoeba castellanii  was selected as a model unicellular eukaryote for this purpose. Homologies of proteins involved in pH and water regulatory mechanisms at cellular levels were compared between humans and A. castellanii. MATERIALS & METHODS: Amino acid sequence homology, structural homology, 3D modeling and docking prediction were done to show the extent of similarities between carbonic anhydrase 1 (CA1), aquaporin (AQP), band-3 protein and H+ pump. Experimental assays were done with acetazolamide (AZM), brinzolamide and mannitol to observe their effects on the trophozoites of  A. castellanii. RESULTS: The human CA1, AQP, band-3 protein and H+-transport proteins revealed similar proteins in Acanthamoeba. Docking showed the binding of AZM on amoebal AQP-like proteins.  Acanthamoeba showed transient shape changes and encystation at differential doses of brinzolamide, mannitol and AZM.  Conclusion: Water and pH regulating adapter proteins in Acanthamoeba and humans show significant homology, these mechanisms evolved early in the primitive unicellular eukaryotes and have remained conserved in multicellular eukaryotes.

Traced on the Timeline: Discovery of Acetylcholine and the Components of the Human Cholinergic System in a Primitive Unicellular Eukaryote Acanthamoeba spp.

Acetylcholine (ACh) is the neurotransmitter of cholinergic signal transduction that affects the target cells via muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors embedded in the cell membrane. Of the cholinergic receptors that bind to ACh, the mAChRs execute several cognitive and metabolic functions in the human central nervous system (CNS). Very little is known about the origins and autocrine/paracrine roles of the ACh in primitive life forms. With the recent report of the evidence of an ACh binding mAChR1 like receptor in Acanthamoeba spp., it was tempting to investigate the origin and functional roles of cholinergic G-Protein coupled receptors (GPCRs) in the biology of eukaryotes. We inferred the presence of ACh, its synthetic, degradation system, and a signal transduction pathway in an approximately ∼2.0 billion year old primitive eukaryotic cell Acanthamoeba castellanii. Bioinformatics analysis, ligand binding prediction, and docking methods were used to establish the origins of enzymes involved in the synthesis and degradation of ACh. Notably, we provide evidence of the presence of ACh in A. castellanii by colorimetric analysis, which to date is the only report of its presence in this primitive unicellular eukaryote. We show the evidence for the presence of homology of evolutionary conserved key enzymes of the cholinergic system like choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in A. castellanii spp., which were found to be near identical to their human counterparts. Tracing the origin, functions of ACh, and primeval mAChRs in primitive eukaryotic cells has the potential of uncovering covert cholinergic pathways that can be extended to humans in order to understand the states of cholinergic deficiency in neurodegenerative diseases (ND).

Sarcomatoid Renal Cell Carcinoma in an Adolescent with Sickle Cell Anaemia.

Malignancies have been reported to occur in people with sickle cell disease. Renal medullary carcinoma (RMC), also tagged seventh sickle cell nephropathy, is an aggressive cancer seen almost exclusively in people with sickle cell disease with more than 160 cases reported worldwide, but only few cases were reported in patients with sickle cell anaemia (HBSS) and from Nigeria. Sarcomatoid renal cell carcinoma is a renal tumour of any histologic variant containing foci of high-grade malignant spindle cells. We report an adolescent girl with sickle cell anaemia (HBSS) who presented with left renal tumour, histology of which confirmed a diagnosis of sarcomatoid renal cell carcinoma (sRCC). Surgical debulking and palliative care with chemotherapy were given, and she demised 10 months after. The rarity of the case and challenges of managing a cancer in the background of a chronic haematologic disorder are highlighted.

Is biological repair of heart on the horizon?

The stem cells keep us young by endogenously repairing us upon need. They do so bysmartly one step forward towards differentiation while another step backward to nurturethe undifferentiated stem cells. They are building blocks for every organ witha differential rate of repair of worn out tissues. Since stem cells can be cultured with a normal karyo type, they could be the ideal source for heart repair after myocardial infarction. As opposed to lower vertebrates and neonatal mice, cardiac regeneration in adult mammalian heart seems to be difficult to assess with a solid evidence of cytokinesis. It becomes more difficult to quantify the level of regeneration after myocardial infarction injury against a background of a large invasion of proliferating inflammatory cells. The question to beraised is how the renewal of a piece of myocardium follows the time line of picking upcell types in series: cardiomyocytes, endothelial cells, smooth muscle cells, fibroblast, pacemaker cells, conducting and Purkinje cells to bring the orchestration of rhythmically contracting and relaxing heart. This review focuses on where we are onthe status of heart repair and cardiac regeneration.

Bioinformatic Insights on Target Receptors of Amiodarone in Human and Acanthamoeba castellanii.

BACKGROUND: Amiodarone is prescribed for certain cardiac arrhythmias in current medical practice. The drug targets and inhibits voltage dependent sodium (Na+ v), calcium (Ca+2 v), potassium (K+ v) channels, enzymes like cytochrome P450 and oxidosqualene cyclase. Past studies have shown that amiodarone exerts antiparasitic effects against Trypanosoma cruzi and Acanthamoeba castellanii. OBJECTIVES: The presence of aforementioned targets and the type of cell death induced by amiodarone in pathogenic eukaryotes like Acanthamoeba castellanii remains to be established. We inferred the presence of homologous targets of amiodarone in A. castellanii compared with humans. METHODS: This study used bioinformatics exploration for amino acid sequence homology, ligand binding attribute predictions, 3D structural model development, and experimental assays that highlight similarity between certain target proteins in Acanthamoeba as compared to humans. RESULTS: The sequence identity scores for amino acids and 3D models show that A. castellanii expresses similar types of targets of amiodarone like Na+ v - K+1 v channels, cytochrome P450 3A4, and lanosterol synthase (oxidosqualene cyclase). We show that even though human like L-type and two pore Ca+2 channels are present in A. castellanii, there was no evidence of the expression of T-type voltage dependent Ca+2 channels. Growth assays showed amoebicidal and amoebistatic effects at doses of 40-80µg/ml. CONCLUSION: The existing bioinformatics tools, ligand binding attribute prediction, and model building offer a specific method to establish homology of proteins, discover drug targets, and facilitate the investigation of the evolution of several types of cardinal ion channels from unicellular eukaryotes to multicellular species as humans.

Antibiotic Effects of Loperamide: Homology of Human Targets of Loperamide with Targets in Acanthamoeba spp.

BACKGROUND: Loperamide is an anti-diarrheal drug prescribed for non-infectious diarrhea. The drug is an opioid receptor agonist, blocker of voltage-dependent calcium channel (Cav) and calmodulin (CaM) inhibitor on human cells. Loperamide has been reported to exert anti-amoebic effects against pathogenic strains of Acanthamoeba castellanii. OBJECTIVES: The precise mode of antibiotic action, cellular target homology with human counterparts and the pattern of cell death induced by loperamide in Acanthamoeba castellanii remain to be established. Additionally, we attempt to establish the presence a primitive Cav in Acanthamoeba castellanii. METHODS: Bioinformatics, 3D structural modelling, ligand binding predictions and apoptotic/ amoebicidal assays were used in this study to answer the above queries. Amino acid sequences and structural models were compared between human and A. castellanii proteins that are involved in the regulation of calcium (Ca+2) homeostasis. RESULTS: Our results show that A. castellanii expresses similar, to near identical types of primitive calcium channels Cav Ac and CaM that are well known targets of loperamide in humans. The growth assays showed anti-amoebic effects of loperamide at different doses, both alone and in combinations with other Ca+2- CaM inhibitors. The synergistic actions of loperamide with haloperidol showed to be more amoebicidal than when either of them used alone. Imaging with Annexin V, Acridine orange and Propidium iodide showed apoptosis in A. castellanii at a dose of 100 µg/ml and necrosis at higher doses of 250 µg/ml. CONCLUSION: Though, Acanthamoeba does not express a homolog of the human mu-opioid receptor, but does shows evidence of the homologs for other known human targets of loperamide that are involved in Ca+2 uptake and Ca+2 signal transduction pathways. This suggests optimization of similar drug interactions with these targets may be useful in developing new approaches to control the growth of this parasite and possibly the diseases caused by it.

Evidence of a M1-muscarinic GPCR homolog in unicellular eukaryotes: featuring Acanthamoeba spp bioinformatics 3D-modelling and experimentations.

Acetylcholine affects the target cellular function via muscarinic and nicotinic cholinergic receptors that are seen to exist in humans. Both the cholinergic receptors are G-protein coupled receptors (GPCRs) that perform cardinal functions in humans. Anti-muscarinic drugs, particularly the ones that target M1 subtype (mAChR1), have consistently shown to kill unicellular pathogenic eukaryotes like Acanthamoeba spp. As the M1 receptor subtype has not been reported to be expressed in the above protist, the presence of an ancient form of the M1 muscarinic receptor was inferred. Bioinformatic tools and experimental assays were performed to establish the presence of a ligand-binding site. A search for sequence homology of amino acids of human M1 receptor failed to uncover an equivalent ligand-binding site on Acanthamoeba, but structural bioinformatics showed a hypothetical protein L8HIA6 to be a receptor homolog of the human mAChR1. Immunostaining with an anti-mAChR1 antibody showed cellular staining. Growth assays showed proliferation and lethal effects of exposure to mAChR1 agonist and antagonist respectively. With the recent authentication of human mAChR1 structure and its addition to the database, it was possible to discover its structural analog in Acanthamoeba; which could explain the effects of anticholinergics observed in the past on Acanthamoeba spp. The discovery of a receptor homolog of human mAChR1 on Acanthamoeba with future studies planned to show its expression and binding to cholinergic agonist and antagonist would help clarify its role in the biology of this protist pathogen.

Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

A New Model of Master of Philosophy in Physiological Sciences.

The objectives of Master of Philosophy (MPhil) in Physiological Sciences are: 1) to describe the new ways in which anatomy, biochemistry and physiology on one hand, and microbiology, pathology and pharmacology on other hand meet their functional requirements through multidisciplinary integrated concepts; 2) to elucidate relationships between cell biology, molecular biology and molecular genetics by connecting dots of how cell functions are driven by molecules and being controlled by genes. This forms the basis of cell, molecular and genetics [CMG] module upon which 7 multidisciplinary modules of Physiological Sciences follow; 3) these 24 credit hours provide the physiological basis for PhD studies as well as faculty development to enhance learning abilities of medical student; 4) the modules constitute Cardio- Respiratory Physiological Sciences, GI and Renal Physiological Sciences, Neurosciences, Endo-Reproductive Physiological Sciences.; 5) it has integrated microbiology, pathology and pharmacology in a unique way through CMG of microbes leading to associated pathology and mechanisms of prescribed drugs; 6) it has additional synopsis and thesis friendly course work leading to comprehensive examinations; 7) the year two deals with research work of 6 credit hours leading to defense of thesis; 8) The MPhil in Physiological Sciences is fundamentally different from what is being offered elsewhere. It prepares and offers a good spring board to dovetail PhD studies as well as faculty and institutional development. This is the first study that deals with innovative programmes in research, learning and education in the field of physiological sciences. This broad-based MPhil would make its recipients competent, critical, confident and productive learner. This is a completely unique design of a curriculum that has no comparable examples elsewhere. Our mission is to educate graduate students in the field of Physiological Sciences such that they have a complete grasp over the broad-based integrated concepts of basic health sciences. Upon completion of their education, the students will be able to use the duality of imagination and skepticism. Hence, the students will contribute to their fields by unfolding their creative energy.

Cardiac acceleration as a marker of vagal withdrawal in heart rate control during exercise in humans.

OBJECTIVE: To investigate whether the time rate of change in heart rate i.e. cardiac acceleration, during aerobic exercise in human subjects could be used to differentiate vagal withdrawal from sympathetic stimulation. METHODS: Fifteen male subjects exercised on a bicycle ergometer at 50 Watts (Step 1), then 100 Watts (Step 2), for 2 minutes each. RESULTS: Heart rate (HR) was monitored from a resting value (mean +/- SD) of 80.3 +/- 12.9 to 113.8 +/- 13.6 beats min-1 in Step 1. In Step 2 exercise, HR increased from 113.8 +/- 13.6 to 145 +/- 20 beats min-1. At the initiation of Step 1, a rapid acceleration of HR was observed in the form of an overshoot response. In contrast to Step 1, a small overshoot response of cardiac acceleration was observed during Step 2. The difference between the mean cardiac acceleration at 10 seconds in Steps 1 and 2 was significant (2.40 +/- 0.19 and 0.71 +/- 0.12 beats min-1 sec-1, p<0.0001). CONCLUSION: The initial vagal withdrawal of exercise-induced tachycardia, as a frontline adaptive mechanism, can be indirectly identified from HR transients using cardiac acceleration as a new marker.

A hospital based preliminary report on sleep disordered breathing in Pakistani population.

BACKGROUND: Sleep Disordered Breathing is an important medical condition leading to significant morbidity and mortality. Western studies have shown its prevalence in middle age to be 9% in women and 24% in men. The aim of this study was to have a preliminary assessment on the frequency of Sleep Disordered Breathing in Pakistani subjects. METHOD: Patients attending a medical clinic were surveyed. A sleep questionnaire elicited information about snoring, witnessed apnea, nocturnal choking and excessive daytime sleepiness. Data were recorded for age, height, collar size and weight. Epworth Sleepiness Scale was used to assess excessive daytime sleepiness. Statistical analysis was by chi square test, t-test and Fisher's exact test. A p-value less than 0.05 was considered significant. RESULTS: A total of 123 subjects (M:F = 2:1) were included in the study. The frequency of snoring was found to be 46%. Snoring with apnoea was reported in 7% and snoring with apnoea and excessive daytime sleepiness in 3%. Snorers were more obese (p < 0.001), older (p < 0.003), with higher body mass index (p < 0.001) and larger collar size (p < 0.006) than non-snorers. Hypertension was more common in patients with sleep disordered breathing (35%) as compared to those without (16%). CONCLUSIONS: This is the first preliminary data on sleep disordered breathing from Pakistan that reflects that the prevalence is similar to that seen in the West. The risk factors and association with hypertension are also comparable.