Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen

The possibility of the existence of a covalent enzyme-phosphoryl intermediate, E-PO3, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase (PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22% of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be rate-limiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 32PO3 group of [gamma-32P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has been determined over the range pH 3-8; a feature with a pKa of approximately 6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a two-stem pseudo Uni Bi mechanism that utilizes a covalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.     

Control of human T-lymphocyte interleukin-2 production by a cAMP-dependent pathway

Cyclic AMP has long been proposed to be the intracellular second messenger that conveys the inhibitory signal for T-cell activation and clonal T-cell proliferation. The present study further explores the mechanism by which the cAMP pathway regulates human T-lymphocyte interleukin-2 (IL-2) production and T-cell blastogenesis. Activation of adenylate cyclase, inhibition of cAMP-dependent phosphodiesterase, or the direct addition of the cell-permeable cAMP analog, 8-N3-cAMP, increased occupancy of intracellular cAMP receptors, inhibited IL-2 production, and reduced T-cell proliferation. However, inhibition of cAMP-dependent protein phosphorylation by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, partially restored IL-2 production. Our data support the conclusion that the cAMP pathway conveys an inhibitory signal for IL-2 production and T-cell proliferation via an integral protein phosphorylation step.     

Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.     

Wheat proteins improve cryopreservation of rat hepatocytes

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze–thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance‐associated proteins such as WCS120, TaTIL, WCS19, and TaIRI‐2. These improved procedures enhance long‐term storage (2–12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77–93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non‐toxic, natural products that do not require animal serum, and are economical and easy to prepare. Biotechnol. Bioeng. 2009;103: 582–591. © 2009 Wiley Periodicals, Inc.     

Maternal body mass index and risk of testicular cancer in male offspring: A systematic review and meta-analysis

Objectives: To date a number of studies have examined the association between maternal weight and testicular cancer risk although results have been largely inconsistent. This systematic review and meta-analysis investigated the nature of this association. Methods: Search strategies were conducted in Ovid Medline (1950–2009), Embase (1980–2009), Web of Science (1970–2009), and CINAHL (1937–2009) using keywords for maternal weight (BMI) and testicular cancer. Results: The literature search produced 1689 hits from which 63 papers were extracted. Only 7 studies met the pre-defined criteria. Random effects meta-analyses were conducted. The combined unadjusted OR (95% CI) of testicular cancer in the highest reported category of maternal BMI compared with the moderate maternal BMI was 0.82 (0.65–1.02). The Cochran's Q P value was 0.82 and the corresponding I² was 0%, both indicating very little variability among studies. The combined unadjusted OR (95% CI) for testicular cancer risk in the lowest reported category of maternal BMI compared to a moderate maternal BMI category was 0.88 (0.65–1.20). The Cochran's Q P value was 0.05 and the corresponding I² was 54%, indicating evidence of statistical heterogeneity. The combined unadjusted OR (95% CI) of testicular cancer risk per unit increase in maternal BMI was 1.01 (0.97–1.06). The Cochran's Q test had a P value of 0.05 and the corresponding I² was 55% indicating evidence of statistical heterogeneity. Conclusion: This meta-analysis, which included a small number of studies, showed that a higher maternal weight does not increase the risk of testicular cancer in male offspring. Though an inverse association between high maternal BMI and testicular cancer risk was detected, it was not statistically significant. Further primary studies with adjustment for appropriate confounders are required.     

Influence of body and genital morphology on relative male fertilization success in oriental beetle

Although the frequently large variance in relative male fertilizationsuccess when females are mated by more than 1 male has beenappreciated for some time, the factors that influence relativepaternity are still poorly understood. Recently, experimentalevidence that morphology of male genitalia influences fertilizationsuccess has been documented in 2 water striders, a dung beetle,and a leaf beetle. We explored the role of male genital morphologyin postcopulatory sexual selection in the oriental beetle. Wemated females to 2 males in succession and assessed relativepaternity by the sterile male technique. Morphology of the malegenitalia was found to strongly influence relative paternitybut only for the first male to mate. Male body size influencedrelative fertilization success as well, but again, only forthe first male; surprisingly, smaller males achieved higherpaternity when mating first. We also found suggestive evidencethat copula duration of both the first and second male to mateinfluenced paternity. Other factors, including female size anddegree of asymmetry of hind tibiae length of males had no effecton relative fertilization success. Our results for the orientalbeetle are novel among sperm precedence studies for 2 reasons:1) traits of the first male appear to be more important in influencingpaternity than those of the second, and 2) smaller, not larger,males achieved greater relative paternity. Our results alsocontribute to the growing body of empirical evidence in supportof the hypothesis that male genitalia evolve by postcopulatorysexual selection.     

CheZ phosphatase localizes to chemoreceptor patches via CheA-short

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a DeltacheZ strain. Localization was observed in wild-type, DeltacheZ, DeltacheYZ, and DeltacheRB cells but not in cells with cheA, cheW, or all chemoreceptor genes except aer deleted. Cells making only CheA-short (CheA(S)) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA(S). Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA(S) while leaving other activities of CheZ intact.     

Degeneration of primary afferent terminals following brachial plexus extensive avulsion injury in rats

Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit beta (TCbeta). Other subtypes weres labeled with the lectin Griffonia simplicifolia 1B4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord,, deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions.