A Functional Truncated Form of c-kit Tyrosine Kinase Is Produced Specifically in the Testis of the Mouse But Not the Rat, Pig, or Human

The complete nucleotide sequence of mouse-truncated mRNA of c-kit, tr-kit, has been determined using the CD1 strain. In this study, the nucleotide sequences of tr-kit from AKR/N, C57BL/6, and ICR strains of mice were determined and found to be identical, although many silent variations were found compared with the sequence in a database for CD1. Tr-kit protein consists of 12 amino acids encoded by the 16th intron and the following 190 amino acids of c-kit. In the sequences of tr-kit encoding 12 specific amino acids, no substitution was detected among the three strains and CD1. Furthermore, RT-PCR analysis clearly showed that tr-kit mRNA expression was present only in testis. No nucleotide mutation in two important regions of the presumptive promoter for tr-kit mRNA was detected within the 16th intron of the mouse strains examined. However, no functional form of tr-kit was found in the rat, pig, or human by sequence analysis and homology testing.     

Nanometer localization of single green fluorescent proteins: evidence that myosin V walks hand-over-hand via telemark configuration

Myosin V is a homodimeric motor protein involved in trafficking of vesicles in the cell. It walks bipedally along actin filaments, moving cargo approximately 37 nm per step. We have measured the step size of individual myosin heads by fusing an enhanced green fluorescent protein (eGFP) to the N-terminus of one head of the myosin dimer and following the motion with nanometer precision and subsecond resolution. We find the average step size to be 74.1 nm with 9.4 nm (SD) and 0.3 nm (SE). Our measurements demonstrate nanometer localization of single eGFPs, confirm the hand-over-hand model of myosin V procession, and when combined with previous data, suggest that there is a kink in the leading lever arm in the waiting state of myosin V. This kink, or "telemark skier" configuration, may cause strain, which, when released, leads to the powerstroke of myosin, throwing the rear head forward and leading to unidirectional motion.     

Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells

How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle selection") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively select the ER Ca2+ store, whereas cholecystokinin additionally recruits a lysosome-related organelle. Similarly, in a pancreatic beta cell line MIN6, acetylcholine selects only the ER, whereas glucose mobilizes Ca2+ from a lysosome-related organelle. We also show that the key to organelle selection is the agonist-specific coupling messenger(s) such that the ER is selected by recruitment of inositol 1,4,5-trisphosphate (or cADP-ribose), whereas lysosome-related organelles are selected by NAADP.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Oxygen affinity of hemoglobin regulates O2 consumption,metabolism, and physical activity

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O(2) delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the alpha-globin gene or Presbyterian mutation in the beta-globin gene. The mutant mice showed increased O(2) consumption and CO(2) production in tissue metabolism, suggesting enhanced O(2) delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O(2) consumption, physical activity, and behavior in mice.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.