Serum insulin concentration and arginine tolerance test in the cynomolgus monkey (Macaca fascicularis)

The standard value of serum insulin was determined to be less than 75 microU/ml with ninety-eight female adult cynomolgus monkeys of wild origin. Then, fifteen apparently healthy laboratory-bred female cynomolgus monkeys aged 6-8 years were studied to know the usefulness of the arginine tolerance test (ATT) by measuring blood glucose, insulin and glucagon. Prior to ATT, all animals had been diagnosed as non-diabetic by the intravenous glucose tolerance test (IVGTT). Arginine hydrochloride was infused intravenously at a dose of 0.5 g/kg. BW under anesthesia. According to the standard value of insulin, fifteen animals were divided into two groups, that is, the low (n = 7) and the high (n = 8) value groups. In the low value group, glucose and insulin value did not change significantly after arginine infusion and their responses were similar to those in the control group (saline infused, n = 4). But glucagon markedly increased from 10 to 45 minutes post infusion. In the high value group, glucagon response was similar to that in the low value group, while glucose and insulin values significantly decreased. It is concluded that the pancreatic alpha-cell function (glucagon secretion) can be judged by the ATT in the cynomolgus monkey but the beta-cell function (insulin secretion) can not be diagnosed.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Resorcinol alkyl glucosides as potent tyrosinase inhibitors

Resorcinol alkyl glucosides 7–12 were developed as novel tyrosinase inhibitors based on the structure of rhododendrin. These were synthesized from 2,4-dibenzyloxybenzaldehyde using either the Wittig or the Horner-Wadsworth-Emmons reaction with Koenigs-Knorr glycosylation as key steps. The tyrosinase inhibitory activity of 7–12 increased with the length of the alkyl spacer between resorcinol and glucose. The 50% inhibitory concentration (IC₅₀) of tetradecyl derivative 12 was 0.39?μM, making it the most potent of the compounds synthesized. The IC₅₀ of 8 (3.62?μM) with a propyl spacer was ca 10?times that of 7 (35.9?μM) with an ethyl spacer. This significant activity difference suggests that an interaction between resorcinol alkyl glucoside and tyrosinase may increase remarkably if the length of the alkyl spacer exceeds C₃.     

Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability

The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.     

Enzyme Kinetics of an Alternative Splicing Isoform of Mitochondrial 8‐Oxoguanine DNA Glycosylase,OGG1‐1b,and Compared with the Nuclear OGG1‐1a

Eight alternatively spliced isoforms of human 8‐oxoguanine DNA glycosylase (OGG1) (OGG1‐1a to ‐1c and ‐2a to ‐2e) are registered in the National Center for Biotechnology Information. OGG1(s) in mitochondria have not yet been fully characterized biochemically. In this study, we purified mitochondrial recombinant OGG1‐1b protein and compared its activity with nuclear OGG1‐1a protein. The reaction rate constant (k_g) of the 7,8‐dihydro‐8‐oxoguanine (8‐oxoG) glycosylase activity of OGG1‐1b was 8‐oxoG:C >> 8‐oxoG:T >> 8‐oxoG:G > 8‐oxoG:A (7.96, 0.805, 0.070, and 0.015 min^?1, respectively) and that of the N‐glycosylase/DNA lyase activity (k_gl) of OGG1‐1b was 8‐oxoG:C > 8‐oxoG:T ?8‐oxoG:G >> 8‐oxoG:A (0.286, 0.079, 0.040, and negligible min^?1, respectively). These reaction rate constants were similar to those of OGG1‐1a except for k_gl against 8‐oxoG:A. APEX nuclease 1 was required to promote DNA strand breakage by OGG1‐1b. These results suggest that OGG1‐1b is associated with 8‐oxoG cleavage in human mitochondria and that the mechanism of this repair is similar to that of nuclear OGG1‐1a.     

Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid

Membrane lipids have been implicated to influence the activity of G-protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus, insight gained from rhodopsin signaling may not be simply translated to other nonvisual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin (NT) receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG-, and POPG-nanodiscs. Binding of the agonist NT to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different, and the apparent affinity of Gαq and Gβ(1)γ(1) to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ(1)γ(1) and NT was crucially affected by the lipid type, with exchange rates higher by 1 or 2 orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a nonvisual GPCR modulate the G-protein-coupling step.     

Comparative mapping of crawling-cell morphodynamics in deep learning-based feature space

Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.     

Donor centrosome regulation of initial spindle formation in mouse somatic cell nuclear transfer: roles of gamma-tubulin and nuclear mitotic apparatus protein 1

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 microm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.