Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.     

Effects of complete hypothalamic deafferentation on the estrous phase of follicle-stimulating hormone release in the cyclic rat

We investigated whether neural afferents to the medial basal hypothalamus play an acute role in the estrous phase of FSH release in the 4-day cyclic rat. A cannula was inserted into the right atrium of the heart under brief ether anesthesia during the early afternoon of proestrus for subsequent blood collections and injection of LHRH. In some of the rats, the medial basal hypothalamus was surgically isolated from the rest of the brain with a small knife under brief ether anesthesia between 2000 h and 2130 h of proestrus. Control groups consisted of naive rats which were not treated during the night of proestrus and sham-operated animals in which the knife was lowered to the corpus callosum between 2000 h and 2130 h or proestrus. Rats were bled at 2200 h of proestrus and at 0200 h, 0600 h and 1000 h of estrus for radioimmunoassay of plasma FSH and LH. The plasma FSH levels in all 3 groups between 2200 h of proestrus and 1000 h of estrus were elevated above levels observed in other cannulated rats bled to the onset of the proestrous phase of FSH release at 1400 h of proestrus. There were no statistically significant differences in plasma FSH or LH concentrations at any of the time periods between the 3 groups of serially bled rats. The deafferentation procedure did not appear to impair the pituitary gland's ability to secret gonadotrophins as injection of 50 ng of LHRH after the bleeding at 1000 h of estrus caused substantial elevations in plasma FSH and LH concentrations which were not different between the 3 groups. The results suggest that neural afferents to the medial basal hypothalamus play no acute role in the estrous phase of FSH release in the cyclic rat.     

Restoration of the periovulatory follicle-stimulating hormone surges in sera by luteinizing hormone releasing hormone in phenobarbital-blocked rats.

The periovulatory increases of follicle-stimulating hormone (FSH) in rat sera can be divided into two phases. The first phase consists of a rise and fall during proestrus and the second phase consists of a rise and fall during estrus. The second phase was not blocked by phenobarbital (100 mg/kg BW) injected i.p. between the first and second phases. In contrast, phenobarbital administered prior to the onset of the first phase blocked both phases of increased serum FSH. In phenobarbital-blocked rats, administration of luteinizing hormone releasing hormone (LHRH) during proestrus, either by s.c. injection (10 μg) or by a 3 hr constant-rate i.v. infusion (50 ng/hr), simulated both the proestrous and estrous phases of increased serum FSH. These results indicate that 1) the second phase of the serum FSH rise is itself not susceptible to phenobarbital blockade, 2) a proestrous mechanism susceptible to phenobarbital alteration is necessary for both phases of increased serum FSH to occur, and 3) administration of LHRH to phenobarbital-blocked rats during proestrus restores both phases of FSH release.     

Morphological convergence of shell shape in distantly related scallop species (Mollusca: Pectinidae)

Morphological convergence is a central concept in evolutionary biology, but convergent patterns remain under‐studied in nonvertebrate organisms. Some scallop species exhibit long‐distance swimming, a behaviour whose biomechanical requirements probably generate similar selective regimes. We tested the hypothesis that shell shape similarity in long‐distance swimming species is a result of convergent evolution. Using landmark‐based geometric morphometrics, we quantified shell shape in seven species representing major behavioural habits. All species displayed distinct shell shapes, with the exception of the two long‐distance swimmers, whose shells were indistinguishable. These species also displayed reduced morphological variance relative to other taxa. Finally, a phylogenetic simulation revealed that these species were more similar in their shell shape than was expected under Brownian motion, the model of character evolution that best described changes in shell shape. Together, these findings reveal that convergent evolution of shell shape occurs in scallops, and suggest that selection for shell shape and behaviour may be important in the diversification of the group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 571–584.     

Simple and universal HPLC-UV method to determine cimetidine, ranitidine, famotidine and nizatidine in urine: application to the analysis of ranitidine and its metabolites in human volunteers

A validated, simple and universal HPLC-UV method for the determination of cimetidine, famotidine, nizatidine and ranitidine in human urine is presented. This is the first single HPLC method reported for the analysis of all four H(2) antagonists in human biological samples. This method was also utilized for the analysis of ranitidine and its metabolites in human urine. All calibration curves showed good linear regression (r(2)>0.9960) within test ranges. The method showed good precision and accuracy with overall intra- and inter-day variations of 0.2-13.6% and 0.2-12.1%, respectively. Separation of ranitidine and its metabolites using this assay provided significantly improved resolution, precision and accuracy compared to previously reported methods. The assay was successfully applied to a human volunteer study using ranitidine as the model compound.     

Comparative molecular docking and molecular-dynamic simulation of wild-type- and mutant carboxylesterase with BTA-hydrolase for enhanced binding to plastic

According to the literature review, microbial degradation of polyethylene terephthalate by PETases has been detected effective and eco-friendly. However, the number of microorganisms capable of such feats is limited with some undesirable bioprospecting results. BTA-hydrolase has been already reported capable of degrading polyethylene terephthalate. Therefore, mutation by in silico site-directed mutagenesis means to introduce current isomer of PETase for polyethylene terephthalate degradative capability as a better approach to resolve this issue. This study aimed to use in silico site-directed mutagenesis to convert a carboxylesterase from Archaeoglobus fulgidus to BTA-hydrolase from Thermobifida fusca by replacing six amino acids in specific locations. This work was followed by molecular docking analysis with polyethylene terephthalate and polypropylene to compare their interactions. The best-docked enzyme-substrate complex was further subjected to molecular dynamics simulation to gauge the binding quality of the BTA-hydrolase, wild-type and mutant-carboxylesterase with only polyethylene terephthalate as a substrate. Results of molecular docking revealed lowest binding energy for the wild-type carboxylesterase-polypropylene complex (-7.5 kcal/mol). The root-mean-square deviation value was observed stable for BTA-hydrolase. Meanwhile, root-mean-square fluctuation was assessed with higher fluctuation for the mutated residue Lys178. Consequently, the Rg value for BTA-hydrolase-ligand complex (～1.68 nm) was the lowest compared to the mutant and wild-type carboxylesterase. The collective data conveyed that mutations imparted a minimal change in the ability of the mutant carboxylesterase to bind to polyethylene terephthalate.     

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb' region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.     

Mycobacterium tuberculosis isolates grown under oxygen deprivation invade pulmonary epithelial cells

Mycobacterium tuberculosis has the ability to adapt to and survive under different environmental conditions, including oxygen deprivation. To better understand the pathogenesis of M. tuberculosis, we studied the invasion of human alveolar (A549) and human bronchial (BBM) epithelial cell lines by M. tuberculosis isolates cultured under oxygen deprivation. We used isolates belonging to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and the laboratory strains H37Rv and H37Ra. We determined that: (1) M. tuberculosis bacilli grown under oxygen deprivation invade epithelial cells, (2) the invasion capacity of all 17 isolates differed, and (3) oxygen deprivation influenced the invasion capacity of these isolates. All isolates invaded the A549 more effectively than the BBM cells. Three of the F15/LAM4/KZN isolates, two of which had extensively drug resistance (XDR) profiles, were at least twice as invasive (≥33%) as the most invasive Beijing isolate (15%) (P < 0.05). We conclude that for a more comprehensive understanding of the pathogenesis of M. tuberculosis, studies should include isolates that have been cultured under oxygen deprivation.