Dynamics of myosin light chain phosphorylation at Ser19 and Thr18/Ser19 in smooth muscle cells in culture

Using thespecific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDamyosin light chain (MLC₂₀) atSer¹⁹ and at bothThr¹⁸ andSer¹⁹, respectively, we visualizedthe dynamics of the MLC₂₀phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3)stimulated with PGF₂. In theresting state, the diphosphorylated form was located in the peripheralregion of the cell, such as the leading edge or the adhesion plaque,and the monophosphorylated form was located not only in the peripheralregion but also on a discontinuous fibrillary structure along the longaxis of the cell. After stimulation with 30 µMPGF₂, although localization ofthe monophosphorylated form changed little, the content of thediphosphorylated form increased and the distribution spread along thefibrillary structure to an extent the same as or similar to that of themonophosphorylated form, which colocalized with actin filament bundles.The diphosphorylation of MLC₂₀ wasmore sensitive to protein kinase inhibitors, HA-1077, HA-1100,staurosporine, wortmannin, and ML-9, than was the monophosphorylation.In light of these observations, we propose thatMLC₂₀ diphosphorylation andmonophosphorylation are regulated by different mechanisms.

     

Computational Observation of an Ion Permeation Through a Channel Protein

The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics. A Na⁺ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10⁸10⁹/sec). In this simulation, it was indicated that the ion permeation through the porin channel proceeds by a push-out mechanism, and that Asp113 is an important residue for the channel activity.     

Protective Effect of Administration of Skim Milk on Exogenous and Endogenous Infection in Mice

In order to minimize the denaturation of proteins in milk, normal cow's milk was pasteurized at 61 C for 20 min. The protective effects of the thus prepared skim milk (low-heat skim milk) on exogenous and endogenous infection were examined as compared with conventional skim milk which was pasteurized at 121 C for 2 sec. The antibody titers to Listeria monocytogenes and Escherichia coli of low-heat skim milk were almost equal to that of raw milk, while no antibody was detected in the conventional skim milk. When mice were given low-heat skim milk or conventional skim milk, the incidence of the translocation of orally inoculated Listeria monocytogenes to the spleen was lower in the low-heat skim milk group than that in the conventional skim milk group. The life span of 7 Gy X-ray irradiated mice given low-heat skim milk was significantly prolonged in comparison to that of mice given conventional skim milk. However, there were no differences in the number of bacteria in the feces or IgA production by Peyer's patch cells between the two groups. These results suggest that antibodies in low-heat skim milk, which still have reactivity to exogenous or indigenous bacteria, may contribute to the protective effects against bacterial infection.     

Prolonged survival of mouse epididymal spermatozoa stored at room temperature

Summary: The viability and fertility of isolated mouse epididymal spermatozoa kept for up to 7 days at various temperatures (4°C, 22°C, and 37°C) were determined. Spermatozoa kept for 3 days at 22°C were still active, while those kept at 37°C or 4°C exhibited great reduction in motility within 2 days after isolation. In vitro fertilizing abilities of spermatozoa left for 0, 1, 2, and 3 days at 22°C were 69.2, 32.5, 9.5, and 4.9%, respectively, when the cleavage rate to two‐cell stage was examined. Transfer of two‐cell embryos produced in vitro with spermatozoa left for 1, 2, and 3 days at 22°C resulted in production of fetuses with efficiencies of respectively 30.2, 11.5, and 16.7%, which were lower (63.3%) than that of embryos derived from in vitro fertilization with fresh spermatozoa. These findings indicate that spermatozoa kept for up to 3 days at 22°C can fertilize oocytes, although at relatively low efficiency. genesis 31:147–155, 2001. © 2001 Wiley‐Liss, Inc.     

The biology of the voleClethrionomys rufocanus: A review

The biology of the gray-sided voleClethrionomys rufocanus in Hokkaido, concerning taxonomy, morphology, phylogeny, distribution, and natural history, is reviewed. Applied issues in forest management (damage, control and census) are also mentioned. AlthoughClethrionomys rufocanus of Hokkaido was originally identified as a distinct species,Evotomys (=nowClethrionomys) bedfordiae Thomas, 1905, current literature generally refers to the gray-sided vole of Hokkaido asClethrionomys rufocanus or asC. rufocanus bedfordiae (vernacular name, the Bedford’s red-backed vole). The gray-sided vole is the most common small mammal in Hokkaido. It inhabits open areas as well as forests, and mainly feeds on green plants. The gray-sided vole has a high reproductive potential; litter size: 4–7; gestation period: 18–19 days; maturation age: 30–60 days old. Although spring-born individuals usually attain sexual maturity in their summer/fall of birth, their maturation is sometimes suppressed under high densities. The breeding season is generally from April to October, but with some regional variation.Clethrionomys rufocanus has a rather specialized diet (folivorous), particularly during winter when it feeds on bamboo grass. Many predators specialize on the grey-sided vole in Hokkaido; even the red fox, which is a typical generalist predator, selectively feeds on this vole. Damage by voles’ eating bark used to be sever on forest plantations in Hokkaido. Censuses of small rodents have been carried out for management purpose since 1954.     

Thermal responses in the citrus fruit fly,Dacus tsuneonis: evidence for a pupal diapause

Thermal responses controlling pupariation and adult eclosion in a citrus fruit fly,Dacus tsuneonis (Miyake), were studied to understand the winter biology of this species. When mature larvae were exposed to various temperature conditions, the highest percentage of pupariation was obtained at 15 °C, although the variance at this temperature was greater than at 20 °C or 25 °C. Pupariation occurred most rapidly at 20 °C and an alternating temperature with a mean of 15 °C. At constant 15 °C, pupae failed to emerge as adults. Pupae were characterized by a reduced respiration rate, which is typical of a diapausing pupa. When insects were stored at different temperatures for 45 days after pupariation, and then transferred to 25 °C, adult eclosion occurred earlier when the initial temperature was 10 °C than when it was 5 °C or 15 °C. Adult eclosion occurred most synchronously and pupal mortality was lowest when insects were stored at 15 °C for 90 days before incubation at 25 °C. These results strongly suggest thatD. tsuneonis enters a pupal diapause.     

Enhancement of differentiation induction of mouse myelomonocytic leukemic cells by extracellular ATP

We examined the effect of ATP on the terminal differentiation of mouse myelomonocytic leukemic M1 cells to macrophages. Although ATP alone did not induce M1 cell differentiation, addition of ATP with the differentiation inducer, interleukin 6 (IL-6), enhanced the induction of differentiation by IL-6 about twofold. Comparison among several adenine nucleotides revealed that the order of effectiveness on differentiation enhancement was ATP > ADP > AMP ≥ adenosine. Using reactive blue 2, a P2 receptor antagonist, we confirmed that the effect of ATP on the stimulation of differentiation was mediated through the P2 purinergic receptor. Examination of cytosolic [Ca2+] elevation by ATP and comparison of potency of differentiation enhancement among several ATP analogs demonstrated that the effect of differentiation enhancement was mediated through P2y purinergic receptors expressed on M1 cell surface. Within 3 h of exposure, ATP alone slightly increased the expression of differentiation-related immediate early response genes, c-myc and JunB, and ATP also enhanced the IL-6–induced expression of these genes. Induction of JunB expression by ATP analogs correlated with their potency of differentiation enhancement, which suggested that induction of JunB by ATP is one of signaling pathways involved in the exertion of its differentiation-enhancing effect. © 1994 wiley-Liss, Inc.     

Characterization of Monoclonal Antibodies for Identification of Borrelia japonica,Isolates from Ixodes ovatus

Monoclonal antibodies for identification of Borrelia japonica isolated from tick, Ixodes ovatus and long-tailed shrew, Sorex unguiculatus in Japan and Borrelia related to Lyme disease (Borrelia burgdorferi sensu lato) were prepared and characterized. All isolates belonging to B. japonica and isolates from I. dentatus and cottontail rabbit in North America reacted with MAb O1441b against flagellin which was prepared from immunized mice with strain HO14, type strain of B. japonica, but isolates from I. persulcatus, patient, and wood mouse, Apodemus speciosus ainu, in Japan, and isolates belonging to B. burgdorferi, B. garinii and B. afzelii from North America and Europe did not. Strains used in this study reacted with MAb P62 against common antigen which was prepared from immunized mice with strain NT24 isolated from I. persulcatus in Japan, but B. japonica did not. These MAbs are useful for identification and differentiation of B. japonica and B. burgdorferi sensu lato in Japan.     

Discrimination of thermal sensation in unhomogeneous thermal environments using theory of quantification II

1. 1. One hundred and forty-five thermal-sensation tests by subjects were performed in unhomogeneous thermal environments and the whole body and the lower leg thermal-sensations were discriminated with physical data of the thermal environments using the discrimination analysis and the expanded theory of quantification II which is able to deal with data including quantitative continuous-variables and qualitative category-variables simultaneously.

2. 2. The discrimination of the whole-body thermal-sensations on the basis of the local-body thermal sensations was also done.

3. 3. As a result, it is shown that the discrimination analysis and the expanded theory of quantification II are able to discriminate the thermal sensation in unhomogeneous environments.