The ''second-codon rule'' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.     

Osteological and histological comparison of the development of the interphalangeal intercalary skeletal element between hyloid and ranoid anurans

Some frog species have a unique skeletal element, referred to as the intercalary element (IE), in the joints between the terminal and subterminal phalanges of all digits. IEs are composed of cartilage or connective tissue and have a markedly differ shape than the phalanges. IEs are highly related to the arboreal lifestyle and toe pads. The IE is found only in neobatrachian frogs among anurans, suggesting that it is a novelty of Neobatrachia. IEs are widely distributed among multiple neobatrachian lineages and are found in the suborders Hyloides and Ranoides (the two major clades in Neobatrachia). However, it is unclear whether the IEs found in multiple linages resulted from convergent evolution. Therefore, in this study, we aimed to examine how similar or different the developmental trajectories of the IEs are between Hyloides and Ranoides. To that end, we compared the osteological and histological developmental processes of the IEs of the hyloid frog Dryophytes japonicus and the ranoid frog Zhangixalus schlegelii. Both species shared the same IE-initiation site and level of tissue differentiation around the IE when it began to form in tadpoles, although the IE developments initiated at different stages which were determined by external criteria. These results suggest that similar mechanisms drive IE formation in the digits of both species, supporting the hypothesis that the IEs did not evolve convergently.     

Biological and biochemical studies of African green monkey lymphotropic papovavirus.

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

Use of mycelia as paths for the isolation of contaminant‐degrading bacteria from soil

Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments. In this study, we demonstrate that mycelia can be used for targeted separation and isolation of contaminant‐degrading bacteria from soil. In a ‘proof of concept’ study we developed a novel approach to isolate bacteria from contaminated soil using mycelia of the soil oomycete Pythium ultimum as translocation networks for bacteria and the polycyclic aromatic hydrocarbon naphthalene (NAPH) as selective carbon source. NAPH‐degrading bacterial isolates were affiliated with the genera Xanthomonas, Rhodococcus and Pseudomonas. Except for Rhodococcus the NAPH‐degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays. All steps of the isolation procedures were followed by cultivation‐independent terminal 16S rRNA gene terminal fragment length polymorphism (T‐RFLP) analysis. Interestingly, a high similarity (63%) between both the cultivable NAPH‐degrading migrant and the cultivable parent soil bacterial community profiles was observed. This suggests that mycelial networks generally confer mobility to native, contaminant‐degrading soil bacteria. Targeted, mycelia‐based dispersal hence may have high potential for the isolation of bacteria with biotechnologically useful properties.     

Involvement of intracellular caveolin‐1 distribution in the suppression of antigen‐induced mast cell activation by cationic liposomes

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross‐linking of high‐affinity immunoglobulin E receptor in a time‐ and dose‐dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the inhibition of store‐operated Ca²⁺ entry. Further, we revealed that the mechanism underlying an impaired [Ca²⁺]_i increase is the inhibition of the activation of the phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Yet, how cationic liposomes inhibit the PI3K‐Akt pathway is still unclear. Here, we focused on caveolin‐1, a major component of caveolae, which is reported to be involved in the activation of the PI3K‐Akt pathway in various cell lines. In this study, we showed that caveolin‐1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin‐1 to the plasma membrane and the colocalization of caveolin‐1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.     

Direct neurite-osteoblastic cell communication, as demonstrated by use of an in vitro co-culture system

Using an in vitro co-culture approach comprising cultured murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells, we found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca2+ mobilization and, after a lag period, osteoblastic Ca2+ mobilization. SV did not have any direct effect on the osteoblastic cells in the absence of neurites. The addition of an alpha1-adrenergic receptor (AR) antagonist, prazosin, dose-dependently prevented the osteoblastic activation that resulted as a consequence of neural activation by SV. These results demonstrate that osteoblastic activation occurred as a direct response to neuronal activation, which activation was mediated by alpha1-ARs in the osteoblastic cells.