Retinal cell transplants: how close to clinical application?
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Animals↗
Biomedical subjects
R K Sharma
Publications and source records attributed to R K Sharma.
Role of preoperative donor-specific transfusion and cyclosporine in haplo-identical living related renal transplant recipients.
A prospective randomized trial of use of donor-specific transfusion and cyclosporine given 24 h before operation was performed in living related renal transplant recipients. The benefits, disadvantages and effect on graft and patient outcome was analyzed. Cyclosporine was started 72 h before operation and 48 h before donor-specific transfusion (DST). Fifteen patients received DST while another 15 age- and sex-matched living related renal allograft recipients on similar immunosuppression served as controls. Patient and donor demographics were similar in the two groups. The DST group had significantly fewer rejection episodes than the control group (0.26 vs. 1.1 rejection episode per patient, p < 0.01). There were fewer episodes of acute rejection in the first 3 months posttransplant in the DST group. Hyperresponder recipients (as tested by mixed lymphocyte cultures) also benefitted by DST which significantly reduced the number of acute rejection episodes (0.25 vs. 1 episode per hyperresponder patient, DST vs. control, p < 0.05). The need for dialysis, incidence of infections and other complications were similar in the two groups. Graft function at 3, 6, 9 and 12 months after transplant was significantly better in the DST group (p < 0.05). Graft survival at 1 year in DST group (85.5%) was not statistically different than control (74.8%). In conclusion, DST and cyclosporine given 24 h before live related renal transplantation is effective in improving graft function and reducing the number of acute rejection episodes which could have a beneficial effect on long-term graft survival.
Acute Disease↗
Effect of artificial stimulants on cryopreserved spermatozoa from cancer patients.
PURPOSE: We determined whether cryopreserved sperm samples obtained from cancer patients before treatment respond to artificial motility stimulants and if this response is related to the extent of disease. MATERIALS AND METHODS: Pre-freeze distribution of disease stage in the different types of cancer and the percentage of the population with or without oligospermia before cryopreservation were examined. Cryopreserved semen samples from 17 cancer patients (10 with testicular cancer, 5 with Hodgkin's disease and 2 with other metastatic disease) were examined for a relationship between post-thaw sperm motion characteristics and patient age or status (survived versus died) and type of disease. Motion characteristics (curvilinear velocity, straight line velocity, average path velocity, linearity and amplitude of lateral head displacement) were analyzed on a computer assisted semen analyzer before (time 0), and 30 and 60 minutes after addition of a 2.5 mM. concentration of pentoxifylline and 2-deoxyadenosine. RESULTS: Post-thaw sperm motion characteristics were not correlated with patient age or status, whether they did or did not have oligospermia, or type of cancer. Compared to baseline values, sperm motion characteristics increased significantly after stimulation at time 0 (p < 0.02) and at 60 minutes (p < 0.05). Oligospermic or nonoligospermic specimens responded to the same extent with pentoxifylline and 2-deoxyadenosine. A negative correlation was noted between overall stage, and type of disease and motion characteristics. CONCLUSIONS: Sperm banking should be encouraged at cancer diagnosis regardless of semen quality. Artificial stimulation of sperm motility results in significant improvement in sperm motion characteristics.
Adult↗
Usefulness of the acrobead test in evaluating human acrosome function in fresh and cryopreserved sperm.
PURPOSE: Assays for the acrosome reaction are usually cumbersome and lack reproducibility. Accurate determination of acrosomal status is important in patients diagnosed with male infertility before proceeding with intrauterine insemination or in vitro fertilization. We determined the optimum capacitation time and acrosomal status of fresh semen specimens in normal fertile men with the Acrobead test, and whether the assay could be used to evaluate cryopreserved semen specimens. MATERIALS AND METHODS: Semen samples from 13 normal donors were divided, with half of the fresh ejaculate used for the Acrobead test and half cryopreserved for a minimum of 24 hours in liquid nitrogen before testing. Fresh and frozen specimens were prepared with the swim-up technique. Sperm concentration was adjusted to 4, 2, 1 and 0.5 x 10(6)/ml. in 4 wells of a 96-well tissue culture plate. Ten microl. polyacrylamide beads (1.5 x 10(6)/ml.) coated with anti-CD46 monoclonal antibodies (MH61 beads) were added to each well. The attachment of beads with acrosome reacted spermatozoa was scored after 0, 1, 3, 6 and 24 hours of incubation. Results were graded on a scale of 0 (no bead binding to the sperm) to 4 (complete attachment to the beads). Specimens with scores of at least 2 were considered normal. RESULTS: A score of at least 2 was noted in 3 of 13 fresh specimens (15.3%) at 1, 9 (69.2%) at 3, 11 (84.6%) at 6 and 13 (100%) after 24 hours. However, a significantly greater number of frozen specimens (8 of 13, or 62%) had a score of 2 or more at 1 hour of incubation and 100% bead attachment to sperm occurred at 3 hours. CONCLUSIONS: Our results indicate that in fresh semen specimens an incubation period of 6 to 24 hours can be used to screen individuals who present with normal sperm characteristics but have slow acrosome reactions. Early acrosome reaction observed in cryopreserved specimens indicates that these spermatozoa may have membrane damage and leakage of acrosome contents as a result of the freeze-thaw process. The Acrobead assay is a simple and objective test that can be used at a clinical andrology laboratory to evaluate the acrosomal status of fresh but not frozen human spermatozoa.
Acrosome↗
Morphological & morphometric studies on liver in rats subjected to repetitive heat stress.
Morphological and morphometric studies were conducted under light microscope on paraffin sections (H&E) of liver from controls and experimental rats subjected to repeated heat stress for 4 h/day, at 38 degrees +/- 0.5 degree C (relative humidity 65-82%) for 2-8 consecutive days. The morphometric variables were derived by intersection-point counting method, using simple square lattice test system A100. Twinning of liver cell plates and anisocytosis characterised morphological changes in rats exposed to heat twice. Following five heat exposures, rat liver showed ballooning degeneration of hepatocytes, associated with sinusoidal compression and loss of normal liver cell plate pattern. The degenerative changes in liver parenchyma were more pronounced in rats that underwent eight heat exposures. Despite hepatocytic degeneration, the numerical density of hepatocytes (Nvh) and the volume density of hepatocytes (Vvh) increased with the increase in number of heat exposures, indicating associated heat induced acceleration in hepatocytic proliferation. A successive fall, with increase in the number of heat exposures, in the sinusoidal length density (Jvs) and in the numerical density of Kupffer cells on area (Nak) revealed progressive damage to hepatic-vascular endothelium, which suggest an adverse effect on the immune status of the animals.
Algorithms↗
Artificial stimulation of cryopreserved human spermatozoa by sodium nitroprusside, 2-chloroadenosine, and 2-deoxyadenosine.
OBJECTIVE: We analyzed the effects of sodium nitroprusside (SNP), 2-chloroadenosine (2-CLA), and 2-deoxyadenosine (2-DA) on sperm motility and motion characteristics of cryopreserved human spermatozoa. METHODS: Thawed semen samples from healthy donors (n = 10) were incubated with the stimulants for 0, 30, 60, 120, and 180 min. The final concentrations used were SNP 25, 50, and 100 nM; 2-CLA 12.5, 25, and 50 microM, and 2-DA 0.5, 1, and 2.5 mM. Sperm motility and changes in motion characteristics were measured on a computer-assisted semen analyzer. RESULTS: Compared with control (0 min, no stimulant), a significant improvement in percentage motility was generally seen with all three stimulants even at 180 min. The sperm motility improved at concentrations of 50 and 100 nM for SNP (except at 120 min), 25 and 50 microM for 2-CLA, and at all concentrations of 2-DA. Other sperm motion characteristics increased to varying extent with the stimulants. Of the three stimulants, all concentrations of 2-DA resulted in significant increases in curvilinear velocity, average path velocity, and amplitude of lateral head displacement at 60 min of incubation. CONCLUSIONS: All three stimulants significantly increased percentage motility for extended intervals of time. 2-DA also improved sperm motion characteristics, though these changes were less uniform with 2-CLA. Motility stimulation by these chemicals may be beneficial in the treatment of cryopreserved and oligozoospermic sperm specimens for use in assisted reproduction.
2-Chloroadenosine↗
Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins.
Post-translational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl and nucleosides. Protein phosphorylation and dephosphorylation is one of the most prevalent and best understood modifications employed in cellular regulation. The bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPEDE) can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for Ca2+ and calmodulin (CaM). The phosphorylation of CaMPDE is blocked by Ca2+ and CaM and reversed by the CaM-dependent phosphatase (calcineurin). The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for CaM. Analysis of the complex regulatory properties of CaMPDE has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaMPDE play a key role in the signal coupling phenomenon. The high molecular weight calmodulin binding protein (HMWCaMBP) was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of HMWCBP was higher in the absence of Ca2+/CaM then in the presence of Ca2+/CaM and reversed by the CaM-dependent phosphatase. Recently, it has become apparent that the binding of myristate to proteins is also widespread in eukaryotic cells and viruses and certainly is of great importance to the correct functioning of an organism. Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the attachment of myristate to the amino-terminal glycine residue of various signal transduction proteins. Cardiac tissue express high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated. The subcellular localization of bovine cardiac muscle NMT indicated a majority of the activity was localized in cytoplasm. Under native conditions the enzyme exhibited an apparent molecular mass of 50 kDa. Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that particulate fraction may contain an inhibitory activity towards NMT. Research in our laboratory has been focusing on the covalent modification of proteins and regulation of various signal transduction proteins. This special review is designed to summarize some aspects of the current work on co- and post-translational modification of proteins in cardiac muscle.
Acyltransferases↗
Physiological perspectives of copper.
Living organisms are composed of only 30 elements, nine of which are required in traces (1:20,000), hence called "Trace elements". The family of trace elements comprises of selenium, chromium, manganese, iron, cobalt, copper, zinc, molybdenum and iodine. Each element has specific associations referred as metalloenzymes.
Animals↗
Viability determination of M.leprae: comparison of normal mouse foot pad and bacillary ATP bioluminescence assay.
Correlation between viability assessment by mouse foot pad and ATP bioluminescence was studied in biopsy specimens from multibacillary leprosy cases. Biopsies were processed for inoculation into mouse foot pad and estimation of bacillary ATP levels by bioluminescent assay by earlier established procedures. ATP content as pg/million bacilli was estimated and correlation was assessed with growth in the mouse foot pad. It was observed that when the ATP content was > 36 pg/million bacterial cells, (> 1% probable viables) there was growth in the mouse foot pad from all the specimens. Similar results were observed when the ATP content was in the range of 3.6 to 35.99 pg/million cells (0.1 to 1% probable viables). The positivity rates in the mouse foot pad decreased when the ATP content decreased further. No positive growth in the specimens below 0.04 pg/million bacilli (< 0.001% viable organisms) was observed. These findings show an overall correlation between viability assessed by mouse foot pad and ATP bioluminescence. These observations validate the concept of ATP content of viable unit of M.leprae being in the order of 10(-15) g/live cell which is in the same order of magnitude as a colony forming unit of cultivable mycobacteria.
Adenosine Triphosphate↗
Microdensitometric scanning procedure for quantitative assessment of hybridization of rRNA targeting probes in leprosy.
In order to develop an objective criteria of grading of positivity of hybridization signals of gene probes targeting rRNA, a microdensitometric scanning procedure was standardised. Ribosomal RNA was extracted from the bacilli harvested from biopsies of leprosy cases across the spectrum and blotted on nitro-cellulose membranes. M. leprae specific rRNA targeting oligonucleotide probes were end-labelled and hybridization was done by the technique standardised and published earlier. The autoradiographs were developed and microdensitometric scanning was done by altering different parameters. Positivity was graded in 5 grades and compared with visual positivity. Microdensitometric scanning procedure and 5 grade system appear to be useful and reproducible. Signals in paucibacillary specimens were in 2+ to 3+ grading range whereas those in multibacillary specimens varied in grades from 2+ to 5+. This approach appears to have potential usefulness for assessing the bacillary load (possibly viable) in the clinical specimens from leprosy cases.
Autoradiography↗
Atropine and testosterone propionate induced atretic changes in granulosa cells of house rat (Rattus rattus) ovary.
Degenerative changes in membrana granulosa of ovaries in R. rattus have been studied using scanning electron microscopy. Ovaries from rats treated with atropine (300 mg/kg body weight) and testosterone propionate (10 IU) were used to study sequential course of atresia in granulosa cells. Granulosa cells undergoing atresia showed degenerative changes in following order i) loosening of intercellular matrix, ii) changed morphology and texture of secretory granules, iii) destabilization of granulosa cell membranes, iv) erosion of cell membrane, v) formation of specific degenerative belts, vi) pycnosis, vii) ghost cell formation and their subsequent mixing in hazzy follicular fluid of cyst. Phenomenon of atresia, its duration, course and underlying causes have been discussed.
Animals↗
Distinct inhibitory ATP-regulated modulatory domain (ARMi) in membrane guanylate cyclases.
Depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the signal transduction activities of the natriuretic factor receptor guanylate cyclases, ANF-RGC and CNP-RGC: there is stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. A defined core ATP-regulated modulatory (ARM) sequence motif within the intracellular 'kinase-like' domain of the cyclases is critical for stimulation, but the mechanism of the inhibitory transduction process is not known. In addition, ATP inhibits the basal cyclase activity of a rod outer segment membrane guanylate cyclase (ROS-GC). The mechanism of this inhibitory transduction process is also not known. These issues have been addressed in the present investigation through a program of deletion mutagenesis/expression studies of the cyclases. The study shows that the ATP-mediated inhibitory transduction processes of the natriuretic factor receptor cyclases and of ROS-GC are identical. The ATP-regulated inhibitory domain of all these cyclases resides within the C-terminal segment of the cyclase. This domain is in a different location from the one representing the ATP-stimulatory ARM. The identification of the inhibitory domain in the C-terminal segment of the cyclase indicates that this segment is composed of two separate domains: one representing a catalytic cyclase domain and the other an ATP-regulated inhibitory (ARMi) domain. These findings establish a novel ATP-mediated inhibitory transduction mechanism of the membrane guanylate cyclases which is distinct from that of its counterpart, the stimulatory ATP-mediated hormonal signal transduction mechanism. Thus, they define a new paradigm of guanylate cyclase-linked signal transduction pathways.
Adenosine Triphosphate↗
Calcium modulation of bovine photoreceptor guanylate cyclase.
Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.
Animals↗
Molecular and pharmacological identity of the alpha 2D-adrenergic receptor subtype in bovine retina and its photoreceptors.
The rat cA2-47 gene encodes the pharmacologically defined alpha 2D-adrenergic receptor (alpha 2D-AR) subtype. Previously, the expression of its mRNA was shown in bovine retina by amplification through the reverse transcription-polymerase chain reaction (RT-PCR) of a region corresponding to the rat alpha 2D-AR, amino acid (aa) residues 382-439, indicating the presence of this subtype in this neural tissue. In the present study, the structure of this gene has been probed and the encoded receptor subtype has been characterized in bovine retina and its photoreceptor cells. The deduced aa sequence of the two bovine gene fragments, aa residues 290-375 and aa residues 392-434, demonstrates 77% overall identity with the rat alpha 2D-AR subtype and 80% overall identity with the mouse alpha 2D-AR. The receptor encoded by the bovine gene was expressed in the retina and its photoreceptors with the typical pharmacological characteristics established for the rat alpha 2D-AR subtype: The receptor bound rauwolscine with a KD of 14 nM in the retina and with that of 19 nM in the photoreceptor cells; the binding association rate constant, k+1, for the ligand was 0.012 min-1, the dissociation rate constant, k-1, was 0.14 min-1 and the half-time for dissociation was 5 min. Oxymetazoline displaced the bound [3H]-rauwolscine with an EC50 value of 85 nM, while SK & F 104078, and prazosin displaced the bound [3H]-rauwolscine with the respective IC50 values of 900 nM and 3000 nM. The other alpha 2-AR subtypes -alpha 2A-AR, alpha 2B-AR, alpha 2C-AR-were not detected in the retina and its photoreceptors. Thus, this study shows that the bovine alpha 2D-AR gene is a structural variant of the rat and mouse genes, that the bovine gene encodes the typical pharmacologically defined alpha 2D-AR subtype, that this subtype is present in its exclusive form in the bovine retina and its photoreceptors, where it may be presynaptic in nature.
Adrenergic alpha-Agonists↗
Coenzyme A dependent myristoylation and demyristoylation in the regulation of bovine spleen N-myristoyltransferase.
N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Little is known about the control and regulation of NMT in higher eukaryotes. Bovine spleen N-myristoyltransferase has been purified and characterized [Raju, RVS, Kalra J & Sharma RK (1994) J Biol Chem 269:12080-12083]. The activation of bovine spleen NMT with thiol reducing compounds, and its inhibition by the oxidizing agent sodium iodate, suggest a role for oxidation/reduction in NMT regulation. Available knowledge concerning coenzyme A (CoA), the thiol in the cell, indicated that the agents tested on NMT could also reduce or oxidize CoA. The studies suggested that reduced CoA is the key regulator of NMT activity, while oxidized CoA did not allow NMT to promote myristoylation. Further, the process of myristoylation and demyristoylation may be governed by NMT, depending on the differential concentration of CoA. The process of demyristoylation could be blocked by excess CoA. We therefore hypothesize that the initial event in the regulation of NMT is an increase in cellular CoA concentration which could be coupled to an increase in protein myristoylation. Once the CoA concentration in the cell decreases due to oxidation, the demyristoylation process would be operative.
Acyltransferases↗
Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase.
In contrast to the membrane guanylate cyclases which are stimulated by extracellular ligands, rod outer segment membrane guanylate cyclase (ROS-GC) activity is modulated intracellularly by calcium in two ways: one, where it is inhibited, and the other, where it is stimulated. The former way is linked to the phototransduction, and physiology of the second is unknown. In both ways calcium modulation of the cyclase occurs through the calcium binding proteins: through guanylate cyclase activating proteins (GCAPs) in the case of phototransduction, and through the recently discovered calcium-dependent GCAP (CD-GCAP) in the case of the other way. The kinase-like domain of ROS-GC is critical for the phototransduction-linked process. The present study shows the expression of alpha and beta chains of S100A1-S100B protein in the bovine retina and demonstrates that this protein stimulates ROS-GC activity in a dose-dependent fashion, that the stimulation is calcium dependent with an EC50 of 17 microM, and that the kinase-like domain is not involved in the calcium-modulated cyclase activation. Instead the involved domain resides at the C-terminal segment, between amino acids 731 and 1054. Thus, this S100A1-S100B protein-mediated calcium-modulated signal transduction mechanism is novel. Furthermore, this study provides the molecular understanding of the two transduction processes mediated by the same ROS-GC, one linked to the low and the other to the high calcium levels.
Amino Acid Sequence↗
Distribution of myristoyl-CoA:protein N-myristoyl transferase activity in rabbit intestine.
Myristoyl-CoA:protein N-myristoyl transferase (NMT) attaches the fatty acid, myristate, to the amino-terminal glycine residue of various proteins involved in cellular regulation and/or signal transduction. We report differences in the activity and properties of NMT in New Zealand rabbit small intestine, ascending colon and descending colon. The mucosa of the small intestine, ascending colon and descending colon was assayed for NMT activity using peptides of known myristoylated proteins (pp60src and catalytic subunit of cAMP dependent protein kinase). Total NMT activity per gram tissue was 5-fold higher in the small intestine and 1.5-fold higher in the ascending colon than in the descending colon. Smooth muscle from the colon also contained low levels of NMT activity. NMT activity was 2- to 3-fold higher in the particulate fraction than in the cytosolic fraction of the mucosa in the descending colon. The apparent molecular mass of NMT in the intestine mucosa was 78 kDa.
Acyl Coenzyme A↗
Reduction of oncoprotein transformation in vitro by albumin.
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Acyltransferases↗