Purification and properties of pyridine nucleotide-independent L-lactate dehydrogenase from Polyporus circinatus

Cell extracts of Polyporus circinatus grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by l-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for l-lactate was 5.9 x 10(-4) M and the oxalate inhibition constant was 1.5 x 10(-4) M. The nature of the prosthetic group is discussed.     

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.     

Seasonal changes in the spermatogenic epithelium of adult Japanese macaques (Macaca fuscata fuscata)

A histological study was undertaken to clarify seasonal changes in the spermatogenic epithelium of Japanese macaques. Testicular tissue samples were excised by biopsies from five adult laboratory-maintained males in mating and non-mating seasons. The samples were fixed with Bouin's solution, embedded in paraffin, and stained with PAS and hematoxylin. Microscopic observations on cross-sections of seminiferous tubules revealed that the seminiferous epithelium in the mating season was thicker than in the non-mating season. PAS-stained granules were found in some of the dark A-type spermatogonia, which significantly increased in the non-mating season. Spermatids of the steps preceding the appearance of the acrosomic cap in stages I to III were observed significantly more often than those in the step coinciding with the formation of the acrosomic cap in stage IV. In stage I, the ratio of mature spermatids or spermatozoa to immature spermatids in the mating season was higher than that in the non-mating season. These findings suggest that spermiogenesis, as well as spermatocytogenesis, is inhibited in the non-mating season.     

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution

 Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, ”i”. Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen ”i” was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with ”i” in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus. Accepted: 6 May 1996     

Evidence for two possible glnB-type genes in Herbaspirillum seropedicae.

Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.     

Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor.

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.