Annual changes in testicular development and occurrence of parasperm in the male reproductive organs of fourspine sculpin, Cottus kazika

Annual changes in testicular development and occurrence of parasperm were investigated using 2-year-old male fourspine sculpins Cottus kazika, based on the histological observation of testes. The male reproductive organ of fourspine sculpins comprised a pair of testes and a sperm duct that functioned as a sperm-storage organ. Male maturity was divided into the following periods: spermatogonial proliferation period (September), early spermatogenic period (October), mid-spermatogenic period (November), late spermatogenic period (December and January), functional maturation period (February and March), and recovery period (April to August). Spermatogenesis rapidly progressed from October to January and continued until the functional maturation period. Parasperm formation, which is known in some cottidae species, was observed in fourspine sculpins. Testicular regression of cultured fourspine sculpins progressed slowly during the recovery period when residual parasperm and empty spaces occupied the testis. The parasperm were immotile and oval and slightly concave on one side; additionally, they stained strongly with hematoxylin and PAS. Seminal lobules of the testis were filled with parasperm during the spawning period; in contrast, the sperm duct was filled with eusperm. These findings were observed in both cultured and wild fish. In this study, the functions of parasperm with regard to reproduction in fourspine sculpins are discussed.     

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

The yeast mitochondrial permeability transition is regulated by reactive oxygen species,endogenous Ca2+ and Cpr3, mediating cell death

We investigated the properties of the permeability transition pore (PTP) in Saccharomyces cerevisiae in agar-embedded mitochondria (AEM) and agar-embedded cells (AEC) and its role in yeast death. In AEM, ethanol-induced pore opening, as indicated by the release of calcein and mitochondrial membrane depolarization, can be inhibited by CsA, by Cpr3 deficiency, and by the antioxidant glutathione. Notably, the pore opening is inhibited, when mitochondria are preloaded by EGTA or Fluo3 to chelate matrix Ca²⁺, or are pretreated with 4-Br A23187 to extract matrix Ca²⁺, prior to agar-embedding, or when pore opening is induced in the presence of EGTA; opened pores are re-closed by sequential treatment with CsA, 4-Br A23187 plus EGTA and NADH, indicating endogenous matrix Ca²⁺ involvement. CsA also inhibits the pore opening with low conductance triggered by exogenous Ca²⁺ transport with ETH129. In AEC, the treatment of tert-butylhydroperoxide, a pro-oxidant that triggers transient pore opening in high conductance in AEM, induces yeast death, which is also dependent on CsA and Cpr3. Furthermore, AEMs from mutants lacking three ADP/ATP carrier (AAC) isoforms and with defective ATP synthase dimerization exhibit high and low conductance pore openings with CsA sensitivity, respectively. Collectively, these data show that the yeast PTP is regulated by Cpr3, endogenous matrix Ca²⁺, and reactive oxygen species, and that it is involved in yeast death; furthermore, ATP synthase dimers play a key role in CsA-sensitive pore formation, while AACs are dispensable.     

Molecular breeding of a biotin-hyperproducing Serratia marcescens strain.

We previously reported that an acidomycin-resistant mutant of Serratia marcescens Sr41, SB304, and a mutant that was derived from SB304 and was resistant to a higher concentration of acidomycin, SB412, produced 5 and 20 mg of D-biotin, respectively, per liter of a medium containing sucrose and urea (N. Sakurai, Y. Imai, M. Masuda, S. Komatsubara, and T. Tosa, Appl. Environ. Microbiol. 59:2857-2863, 1993). In order to increase the productivity of D-biotin, the biotin (bio) operons were cloned from strains SB412, SB304, and 8000 (wild-type strain), and pLGM412, pLGM304, and pLGW101, respectively, were obtained through subcloning. These plasmids harbored 7.2-kb DNA fragments coding for the bioABFCD genes on a low-copy-number vector and were introduced into SB304, SB412, and 8000. Among the resulting recombinant strains, SB412(pLGM304) exhibited the highest D-biotin production (200 mg/liter) in the production medium. The plasmid was stably maintained in cells. Unexpectedly, SB412(pLGM412) grew very slowly, and the D-biotin productivity of this recombinant strain was not evaluated because pLGM412 was unstable.     

Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.     

Oxidative modification of glycated low density lipoprotein in the presence of iron

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.