A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side-chains of the Streptococcus mutans rhamnose-glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side-chains. The roles of rgpH and rgpI were evaluated in a rhamnan-synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnan-specific antiserum, co-transformation of this strain with rgpI increased the number of glucose side-chains and decreased immunoreactivity with the rhamnan-specific antiserum significantly. These results suggest that two genes are involved in side-chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the first report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis.     

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic-polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-gamma, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 microM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.     

Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster

To better understand the genetic bases of postmating responses in Drosophila melanogaster females, we screened a collection of P{GS} insertion lines and identified two insertions in sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Stereoselective binding and degradation of sulbenicillin in the presence of human serum albumin

Binding of sulbenicillin (SBPC) isomers to human serum albumin (HSA) was stereoselective. There were at least two classes of binding sites on HSA for SBPC isomers. At the stereoselective high affinity site, binding was in favor of R-SBPC, the binding constant of R-SBPC being approximately 2.3-fold greater than that of S-SBPC. By using site marker ligands, it was revealed that the stereoselective site was Site I (warfarin binding site). Affinity for the low affinity (nonstereoselective) site was similar for the diastereomers, approximately 7--30-fold lower than for the stereoselective site. R-SBPC and S-SBPC appeared to displace each other competitively at both binding sites. On the other hand, R-SBPC was degraded much faster than S-SBPC in the presence of HSA, with a degradation rate constant approximately 7-fold greater for R-SBPC than for S-SBPC. The degradation of R-SBPC was inhibited in the presence of warfarin and dependent on the concentration of R-SBPC bound to Site I. The results demonstrate that Site I is responsible for the stereoselective degradation.     

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP‐evoked production of interleukin‐6 (IL‐6) and its intracellular signals were examined using a mouse microglial cell line, MG‐5. ATP, but not its metabolites, produced IL‐6 in a concentration‐dependent manner. Although ATP activated two mitogen‐activated protein kinases, i.e. p38 and extracellular signal‐regulated protein kinase, only p38 was involved in the IL‐6 induction. However, the activation of p38 was not sufficient for the IL‐6 induction because 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL‐6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL‐6 production by the cells. The ATP‐evoked IL‐6 production was attenuated by Gö6976, an inhibitor of Ca²⁺‐dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX‐insensitive P2Y receptors activates p38 and Ca²⁺‐dependent PKC, thereby resulting in the mRNA expression and release of IL‐6 in MG‐5. This is a novel pathway for the induction of cytokines in microglia.