Srinivas Pentyala

Identification and characterization of a novel protein that regulates RNA–protein interaction

In a previous study [Nachaliel et al., 1993], we identified an RNA‐binding protein (RBP) in FTO‐2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA‐binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near‐homogeneity (Mr ∼25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA‐binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS‐polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ‐1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed. J. Cell. Biochem. 72:435–444, 1999.

Prostasomes : Current concepts

Prostasomes are membranous vesicles secreted by prostate gland, and they contain large amounts of cholesterol, sphingomyelin, calcium, and several enzymes. Prostasomes are involved in a number of biological functions. At ejaculation, these prostasomes are expelled with prostate secretions and are to be found in the seminal plasma as seminal prostasomes, which facilitate sperm function in various ways.

Volatile anesthetics modulate the binding of guanine nucleotides to the α subunits of heterotrimeric GTP binding proteins

The effects of volatile anesthetics on guanine nucleotide binding to the purified alpha subunits of heterotrimeric GTP binding (G) proteins were studied. At sub-anesthetic doses, halothane, isoflurane, enflurane and sevoflurane inhibit exchange of GTPgammaS for GDP bound to Galpha subunits and markedly enhance the dissociation of GTPgammaS, but fail to suppress GDPbetaS release. Nucleotide exchange from non-myristoylated Galpha(i1) is similarly inhibited in the absence of any membrane lipid or detergent. The degrees of inhibition of GDP/GTPgammaS exchange and enhancement of GTPgammaS dissociation are in the same order: alpha(i2)alpha(i1)alpha(i3)alpha(s). By contrast, Galpha(o), which is closely related to Galpha(i), is completely insensitive to anesthetics. We conclude that volatile agents, at clinically relevant doses, have a direct effect on the conformation and stability of the GTP/Mg(2+) bound state of some, but not all Galpha subunits. By destabilizing this state, volatile agents may uncouple metabotropic and other heptahelical receptors from pathways modulating neuronal excitation.

The role of endoluminal ultrasonography in urology: current perspectives

Endoluminal ultrasonography (ELUS) is a noninvasive diagnostic technique used in urology to image tubular structures of the urinary tract. Through advancements in technology, modern ELUS is able to create three-dimensional images, which provide valuable clinical information for the diagnosis and treatment of urologic disorders. The efficiency and accuracy of this technology is confirmed through validation studies using human and animal models. Although a relatively new method, the clinical application of this technique holds great promise in the field of endourology. The technology, advantages, limitations, validation studies, clinical applications, and future of ELUS are explored through this comprehensive review of current urologic literature.

Effects of inhalational anesthetics on α2-adrenergic signaling in isolated platelets

(1) The hypothesis that inhalational anesthetics affect G-protein linked alpha2 adrenergic signaling pathway was investigated using human platelets as a model system. (2) Alpha2 receptor stimulation by UK-14304, a potent and selective agonist, inhibits cAMP production induced by prostaglandin I2 (PGI2). (3) Brief stimulation (30 s) with PGI2 raised cAMP levels in platelets by 25-fold; UK-14304 suppressed the PGI2 stimulus by 80%. (4) Halothane at fractional minimum alveolar concentration (MAC) through super physiological levels (16 MAC) had no effect on basal or prostacyclin stimulated levels of cAMP, nor did it have any effect on the inhibition of cAMP production by UK-14304. Moreover, isoflurane, enflurane and sevoflurane had no significant effect on cAMP production at 1.5 or 8 MAC. The results suggest alpha2 and PGI2 signaling pathways are not sensitive to volatile anesthetics including the alpha2 or PGI2 receptor/G-protein complex, G-protein/adenylyl cyclase complex and adenylyl cyclase itself. (5) The possibility that halothane and related anesthetics act more distally in the pathway, on cAMP-dependent protein kinase (PKA), was investigated by measuring the phosphorylation pattern of endogenous platelet proteins by PKA. (6) An increase in the [32P]phosphate incorporation was observed in platelets exposed to either, low doses of PGI2 or isobutylmethylxanthine (IBMX). Halothane, isoflurane, enflurane or sevoflurane further increased the level of [32P]-incorporation. The apparent increase in PKA activity suggests that at least in platelets, volatile anesthetics activate PKA-dependent pathways which should antagonize alpha2 adrenergic signaling.

A Novel Point-of-Care Biomarker Recognition Method: Validation by Detecting Marker for Diabetic Nephropathy

Biological fluid collection to identify and analyze different disease markers is a routine and normal procedure in health care settings. Body fluids are as varied as urine, blood, mucus, cerebrospinal fluid (CSF), tears, semen, etc. The volumes of the collected fluids range from micro liters (e.g., tears, CSF) to tens and hundreds of milliliters (blood, urine, etc.). In some manifestations, a disease marker (particularly protein markers) can occur in trace amounts, yet the fluids collected are in large volumes. To identify these trace markers, cumbersome methods, expensive instruments, and trained personnel are required. We developed an easy method to rapidly capture, concentrate, and identify protein markers in large volumes of test fluids. This method involves the utilization of two antibodies recognizing two different epitopes of the protein biomarker. Antibody-1 helps to capture and concentrate the biomarker and Antibody-2 adsorbed or conjugated to nanogold beads will detect the biomarker. This method was validated in capturing and detecting lipocalin type prostaglandin-D2 synthase, a marker in urine that implicates diabetic nephropathy. A one-step collection, concentration, and detection device was designed based on this method. This device can replace many of the normal body fluid collection devices such as tubes and containers. A one-step fluid collection and biomarker capture and concentration device for rapid diagnosis of diseases has tremendous advantage in terms of cost and providing timely results.

Evaluation of prostaglandin D2 as a CSF leak marker: implications in safe epidural anesthesia

Background It is accepted that there is a severe risk of dural puncture in epidural anesthesia. Of major concern to anesthesiologists is unintentional spinal block. Reliable identification of cerebrospinal fluid (CSF) from the aspirate is crucial for safe epidural anesthesia. The aim of this study was to determine whether prostaglandin D2 could be clinically used as a marker for the detection of CSF traces. Methods After obtaining Institutional Review Board approval and patient consent, CSF was obtained from patients undergoing spinal anesthesia, and blood, urine, and saliva were obtained from normal subjects and analyzed for prostaglandin D2 (PGD). CSF (n=5) samples were diluted with local anesthetic (bupivacaine), normal saline and blood in the ratios of 1:5 and 1:10. PGD levels in the CSF samples were analyzed with a PGD-Methoxime (MOX) EIA Kit (Cayman Chemicals, MI). This assay is based on the conversion of PGD to a stable derivative, which is analyzed with antiserum specific for PGD-MOX. Results Different concentrations of pure PGD-MOX conjugate were analyzed by EIA and a standard curve was derived. PGD levels in CSF and CSF with diluents were determined and the values were extrapolated onto the standard curve. Our results show a well-defined correlation for the presence of PGD both in straight CSF samples and in diluted CSF (dilution factor of 1:5 and 1:10). Conclusion Prostaglandin D2 was reliably identified in CSF by enzyme-linked immunosorbent assay when diluted with local anesthetic, saline, and serum, and can be used as a marker to identify the presence of CSF in epidural aspirates.

Osteoporosis in Female Athletes

Osteoporosis is a condition that affects almost 10 per-cent of the total female population worldwide and is especially prevalent among female athletes. This dis-ease is characterized by a decrease in bone mass and density and enlargement of bone spaces producing po-rosity and fragility. With the number of women of all ages participating in physical activity steadily increas-ing, the relationship between osteoporosis and female athletes is a growing concern [1-4]. It is estimated that more than 6 million women now compete in strenuous exercise, worldwide. Despite the benefits of exercise, excessive and strenuous physical activity can have neg-ative effects on the reproductive and skeletal systems, leading to osteoporosis [5,6]

Effect of Maitotoxin on Guanine Nucleotide Interaction With G-Protein α Subunits

Maitotoxin is a potent water-soluble polyether toxin produced by the marine dinoflagellate Gambierdiscus toxicus. Although associated with increased calcium uptake, mobilization of internal calcium stores, and enhanced phosphoinositide metabolism, the primary molecular mechanism underlying its actions remains unclear. In this study, we evaluated the effects of maitotoxin (MTX) on the interaction of guanine nucleotides with G-protein α sub-units. Equilibrium binding of the nonhydrolyzable GTP analog, GTPγS, to α subunits (Go, Gs, Gil, Gi2, and Gi3) was decreased in the presence of MTX. Furthermore, reconstitution of Gα with Gβγ dimer showed a reversal of the inhibition elicited by MTX. GDP/GTP exchange rate for Gα subunits was significantly inhibited in the presence of MTX. MTX had no effect on the rate of GDP or GTP dissociation from α subunits. Also, the mastoparan-induced component of nucleotide exchange is not effected by MTX. These results suggest that MTX acts on Gα subunits to modulate their interaction with guanine nucleotides, perhaps by stabilizing an empty state of the α subunit. Accordingly, MTX may disrupt the normal signal transduction pathways by inhibiting GTP binding to Gα subunits and interfering with the GDP/GTP exchange.

The Effectiveness of Hypnosis and Suggestive Techniques in Reducing Postoperative Side Effects in Children

Introduction Suggestive techniques including hypnosis have been used to alleviate surgical side effects [1]. Hypnosis is described as an artificially induced trancelike state, in which the subject is highly susceptible to suggestion, oblivious to all else, and responds readily to the commands of the hypnotist [2]. Hypnosis was used as a therapy throughout many millennia. Since ancient times as old as Mesopotamian, Mayan and Indus Valley civilizations, hypnosis was seen as a cure to many physical and mental illness. Franz Anton Mesmer, a Viennese physician played a key role in the development of hypnosis for modern medical use [3]. While in the early 19th century, several hundred surgical interventions were described with hypnosis as the sole anesthetic, and it was prejudicially abandoned with the onset of the anesthetic age [4]. More recently however, the value of hypnosis has been increasingly recognized in medicine, especially in adults [5, 6]. Hypnosis could also be used on pediatric patients and yield effective results, as children have a vast imagination and lack full cognitive development, thereby making them more susceptible for suggestions [7]. However, literature on the effectiveness of suggestive techniques in children is scant. Using the terms, ‘suggestive techniques’ or ‘hypnosis’ and ‘children’ or ‘pediatric’ and ‘postoperative’, studies from the database – PubMed, managed by US government funded National Center for Biotechnology Information, were collected and reviewed. The quality of the study and the ability to draw conclusions with respect to effect on postoperative pain, hospital stay, anxiety and nausea/vomiting were analyzed. This review summarizes the available literature and draw conclusions for possible postoperative benefit of suggestive techniques in pediatric patients.