Complete Genome Sequence of Streptococcus mutans GS-5, a Serotype c Strain

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.     

Purification and Mass Spectrometric Characterization of Sesbania aculeata (Dhaincha) Stem Lectin

A glucose specific lectin (STA) was isolated from Sesbania aculeata stem by using Sephadex G-50 affinity column chromatography. The lectin is a glycoprotein having 29 kDa subunit molecular weight. Two dimensional gel electrophoresis analysis revealed that the lectin existed in two isomeric forms with varied carbohydrate content as analyzed by high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and N-terminal sequence (LDSLSFTYNNFE) analysis of this lectin showed 95% homology with stem lectin SL-I (accession no. AJ585523) from peanut plant. The nucleotide sequence of the lectin (STA) was submitted to the gene bank (accession no. EU263636).     

Amphiregulin promotes the proliferation of trophoblast cells during preimplantation development of porcine embryos

Our objective was to investigate the effects of in vitro culture (IVC) medium supplemented with amphiregulin (AREG) on the preimplantation embryonic development of porcine (Genus: Sus domestica, Species: Landrace) embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA). In vitro fertilization and PA embryos at the 1-cell stage were cultured in IVC medium supplemented with 0, 0.5, 5, or 50 ng/mL AREG for 7 d. There were significantly greater total numbers of cells in blastocysts of IVF and PA embryos cultured with 50 ng/mL AREG compared with that of controls. In vitro fertilization and PA embryos were then cultured in NCSU-23 medium supplemented with 50 ng/mL AREG on Days 1 through 7, Days 1 through 3 (early stage), or Days 4 through 7 (late stage), or without AREG. There were significantly greater numbers of trophoblast cells in the late-stage and full-term groups of IVF and PA embryos than in the early-stage and control groups. The presence of AREG protein in IVF-derived blastocysts was detected using a polyclonal AREG antibody and indirect immunofluorescence. Amphiregulin protein was localized in both the cytoplasm and nucleus. Using real-time polymerase chain reaction, we detected the expression of AREG mRNA in all developmental stages of IVF and PA embryos; however, the expression level varied according to stage. Thus, the incubation of porcine IVF and PA embryos in AREG-supplemented culture medium mainly at the late preimplantation stage increases the numbers of trophoblast cells.     

Collection and characterization of semen in Mithun (Bos frontalis) bulls

The objective of this study was to collect semen from semiwild Mithun (Bos frontalis) bulls using an artificial vagina (AV) and to determine semen characteristics. Collection of semen with an AV was attempted in five Mithun bulls using both anestrous and estrous Mithun females. No Mithun bull mounted an anestrous female Mithun during 60 trials, but satisfactory mounting, including extension of the penis, occurred in 25 trials with estrous Mithun females. In 15 of these trials, semen was successfully collected in an AV with an internal temperature of 42 to 46 °C. However, in 10 trials with an AV with an internal temperature of 36 to 40 °C, semen was not collected. Mean (± SEM) intervals to first mount and to ejaculation in the AV were 27.9 ± 3.6 sec and 113.8 ± 6.6 sec, respectively. Semen volume and pH were 3.1 ± 0.35 mL and 6.59 ± 0.04, and mean mass activity (scale, 0 to 4), initial sperm motility, live sperm count, sperm concentration, total number of sperm in the ejaculate, and overall sperm length were 2.2 ± 0.3, 78.6 ± 2.6%, 80.7 ± 2.2%, 710.8 ± 66.8 × 10⁶/mL, 2114 ± 364.4 sperm, and 67.9 ± 0.6 μm, respectively. The proportion of morphologically normal sperm was 80.6 ± 0.2%, whereas the proportion with a morphologically abnormal head, midpiece, tail, and acrosome were 4.2 ± 0.4%, 1.6 ± 0.5%, 6.1 ± 1.1%, and 7.1 ± 0.9%, respectively. The mean incidence of tail-less heads and proximal and distal protoplasmic droplets were 0.5 ± 0.1%, 0.3 ± 0.2%, and 2.4 ± 0.3%, respectively. In conclusion, we successfully collected semen from semiwild Mithun bulls with an AV maintained at 42 to 46 °C, and overall, the semen was within the normal range of that collected from fertile domestic bulls.     

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity. Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signal transduction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module: MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C*

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs)⁴ have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of the extracellular domain of mammalian receptor GCs vary (less than ∼15% similarity), as would be expected given the diversity in the ligands that bind to and activate these receptors. The KHD shows ∼25–30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2, 3). The catalytic domains of mammalian receptor GCs are more conserved (∼80% sequence similarity). The gradual increase in sequence similarity across the various domains, with the extracellular domain being the most diverse and the cyclase domains sharing the maximum sequence similarity, is a reflection of the ability of these receptor GCs to converge diverse extracellular signals to a unified output of cGMP production. The guanylyl cyclase domains of receptor GCs can be classified as members of the Class III family of nucleotide cyclases (4). The recent crystal structures of a bacterial guanylyl cyclase (5) and a eukaryotic soluble guanylyl cyclase (6) show similarities in the overall three-dimensional structure of adenylyl and guanylyl cyclases and also highlight the critical residues that determine substrate utilization (either ATP or GTP) in these enzymes.Guanylyl cyclase C (GC-C) serves as the receptor for the guanylin family of endogenous peptides as well as for the exogenous heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria (7, 8). GC-C is predominantly expressed on the apical surface of epithelial cells in the intestine, although robust extra-intestinal expression is observed in the kidney and reproductive tissues of the rat (9–12). The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2, 3).A sequence of ∼70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13). This region is predicted to form an amphipathic α-helix and could also adopt a coiled coil conformation (14, 15). The linker region is also present in soluble (cytosolic) guanylyl cyclases where it connects the N-terminal heme binding regulatory domain to the C-terminal catalytic cyclase domain. The linker region is suggested to act as a dimerization module in receptor GCs (16–18) and has also been implicated in heterodimerization of the α and β subunits of soluble guanylyl cyclases (19, 20). However, there are several reports to the contrary that indicate that the linker does not affect the dimerization of receptor GCs (14, 15). Nevertheless, the critical importance of the linker in regulating the activity of receptor GCs is shown by the fact that mutations in this region of the retinal guanylyl cyclase (RetGC-1) are associated with autosomal dominant cone-rod dystrophy in humans (16, 21). We show here through extensive mutational and biochemical analysis that the linker regions in two receptor GCs, GC-C and guanylyl cyclase A (GC-A), play an important role in repressing the catalytic activity of the receptors in the absence of their ligands. In addition, our results provide for the first time a molecular explanation for detergent-enhanced guanylyl cyclase activity in this family of receptors and suggest a mechanism for this activation that could involve a hydrophobic interaction between the linker region and the guanylyl cyclase domain.     

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24 h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.     

Maintenance of brain thyroid hormone level during peripheral hypothyroid condition in adult rat

Thyroid hormones are essential for normal functioning of adult mammalian brain. The present investigation deals with the understanding of the time course of thyroid hormone homeostasis in adult rat brain. Animals were rendered hypothyroid by PTU injections (2 mg/100 g bw) for 30 consecutive days. Serum and synaptosomal T3/T4 content, synaptosomal AChE and Na+-K+-ATPase activities were determined on alternate days. While serum T4 level initially increased on the second day compared to control, serum T3 declined in a triphasic pattern; the first phase lasting from the second day to the 6th day, the second phase ended on the 14th day and last phase continued till the 30th day. Cerebro-cortical synaptosomal T3 level increased on the 2nd day from the control, attained a peak on the 4th day, remained stable until the 18th day, and abruptly declined on the 20th day. Synaptosomal T4 content remained negligible or undetected throughout. Synaptosomal membrane Na+-K+-ATPase and AChE activity exhibited an inverse relationship during the experimental regime, being much more prominent on the 2nd, 18th and 20th day coinciding with the variations in brain T3 level. Thus, the study identifies the onset of central homeostasis between the first and second day, its continuation for about 16-18 days and its termination between the 18th and 20th day.