Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus

Summary.  We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK- gag was confirmed to express the FIV Gag precursor protein in the cells. Accepted November 12, 1997 Received August 26, 1997     

Proliferative activity of early hemopoietic precursors under conditions of increased and decreased activity of protein kinase C in mouse bone marrow

Activation of protein kinase C (transfer from cytosol to cell membranes) with phorbol ester increases proliferation of splenic colony-forming units in the bone marrow. Subsequent incubation of bone marrow cells with the factor inhibiting proliferative activity of early hemopoietic precursors results in the recovery of protein kinase C activity in cell cytosol and a decrease in proliferative activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 541–543, November, 1997     

Genetic heterogeneity of heat shock protein synthesis and sensitivity of mouse splenocytes to antiproliferative effect of alkylating agents

The intensity of heat shock protein synthesis by splenocytes is assessed in mice of two strains with genetically different sensitivity to antiproliferative effect of alkylating effect of maphosphamide. The content andde novo production of heat shock proteins in resistant BALB/c mice are higher than in sensitive DBA/2 mice. Exposure to heat shock increases cell resistance to antiproliferative action of maphosphamide. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 561–565, November, 1997     

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet β-cells

 Intracellular cAMP-dependent modulation of L-type Ca²⁺ channel activation in cultured rat islet β-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca²⁺ current (I _Ca) showed a fast activation followed by a slow inactivation, and was sensitive to Ca²⁺ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak I _Ca in a concentration-dependent manner. Values of the half-activation potentials (V _1/2), taken from activation curves for I _Ca, were –16.7 ± 1.8 and –21.9 ± 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet β-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca²⁺ channels. Received: 9 September 1997 / Received after revision: 19 November 1997 / Accepted: 21 November 1997     

Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CREA protein

The pr1 gene of the entomopathogenic fungus Metarhizium anisopliae encodes a serine protease that is highly active towards the insect cuticle and whose synthesis is subject to both carbon and nitrogen repression. The pr1 promoter region was sequenced revealing the presence of putative CREA- and AREA-binding sites. In vitro bandshift experiments demonstrated that an Aspergillus nidulans GST-CREA fusion protein was capable of binding to two of the three putative CREA sites. Using a PCR-based strategy the M. anisopliae crr1 gene was identified; it encodes a putative C₂H₂-type DNA-binding protein with significant sequence similarity to A. nidulans CREA. Complementation experiments with an A. nidulans strain carrying creA204 demonstrated that CRR1 can partially substitute for CREA function. Received: 29 November 1996 / 4 February 1997     

Yeast bcy1 mutants with stationary phase-specific defects

Entry into the stationary phase requires the yeast BCY1 gene, which encodes the regulatory subunit of the cAMP-dependent protein kinase (cAPK). New bcy1 mutants, constructed by in vitro mutagenesis of the 3′-region encoding the cAMP-binding domains, were classified as early or late-acting mutants based on viability studies. The late-acting bcy1 mutants accumulated fewer stationary phase-specific Bcy1p isoforms and had decreased cAPK activity. This late-acting class is novel and dies after 7 days in culture, later than two previously reported stationary phase mutants, ubi4 and ard1. Received: 4 November 1996 / 12 April 1997     

Detection of bovine immunodeficiency-like virus infection in experimentally infected calves

Summary.  Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described. Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome. An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection. Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)₆p26) expressed in E. coli and purified by metal affinity chromatography, in ELISA and Western blot studies. The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up. Accepted August 14, 1997 Received November 28, 1996     

Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80–84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radio-labeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited. Received: 9 July 1997 / Accepted: 3 November 1997     

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida

Summary.  With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750 – 800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a M_r of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3′ end of the viral genome encompassing the 3′ non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3′ non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested. Accepted November 5, 1997 Received July 25, 1997     

Aromatic residues affecting permeation and gating in dSlo BK channels

 Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site. Received: 16 September 1997 / Received after revision: 20 November 1997 / Accepted: 21 November 1997     

Over-expression of the yeast BFR2 gene partially suppresses the growth defects induced by Brefeldin A and by four ER-to-Golgi mutations

The fungal metabolite Brefeldin A (BFA) disrupts the Golgi apparatus and its incoming protein flux. We developed a genetic approach to identify yeast proteins involved in the protein transport step that BFA blocks. The BFR2 gene (YDR299W) was thus isolated as a high-copy suppressor of the growth defects induced by BFA in a sensitive strain of Saccharomyces cerevisiae. Although BFR2 over-expression did not cause a secretory block or slow-down, it partially suppressed the growth defect of four mutants blocked at the step of budding or docking of small vesicles en route to the Golgi (sec13-1, sec16-2, sec23-1, ypt1-1). The essential BFR2 gene was predicted to encode an extremely hydrophilic product containing two short regions with potential coiled-coils, one of which corresponds to a cluster of acidic residues. Received: 10 September / 3 November 1997     

RPG1: an essential gene of Saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G₁ phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or α-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G₁ cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G₁ at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G₁ phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3. Received: 6 October 1997 / 8 November 1997     

Nucleotide sequence analysis of the coat protein genes of two Korean isolates of sweet potato feathery mottle potyvirus

Summary.  The coat protein (CP) genes of the genomic RNA of two Korean isolates of sweet potato feathery mottle potyvirus (SPFMV), SPFMV-K1 and SPFMV-K2, were cloned and their complete nucleotide sequences were determined. Sequence comparisons of the two Korean isolates showed 97.8% amino acid identity in the CP cistron, and 79.9% to 99.0% identity with those of 6 other known SPFMV strains. Of 74 amino acid changes totally among the SPFMV strains, 39 changes were located at the N-terminal region. Pairwise amino acid sequence comparison revealed sequence similarities of 48.6 to 70.2% between SPFMV and 20 other potyviruses, indicating SPFMV to be a quite distinct species. Multiple alignment of the CP cistrons from other potyviruses showed that most of the conserved amino acid residues of the genus Potyvirus are well preserved in the corresponding locations. Accepted November 13, 1997 Received September 1, 1997     

Cultivation of bloodstream forms of Trypanosoma carassii, a common parasite of freshwater fish

Trypanosoma carassii (syn. T. danilewskyi) is a widespread parasite of carp and other cyprinid as well as some noncyprinid freshwater fish. It lives extracellularly in the blood and tissues of its hosts, causing chronic infections. In this paper the isolation of T. carassii from fish blood and the propagation and cloning of bloodstream forms in vitro are described. By several criteria, cultured and fish-derived trypomastigotes are indistinguishable. The culture system should be useful for the biochemical characterization of this trypanosome and its interaction with the fish immune system. Received: 10 September 1997 / Accepted: 26 November 1997     

Seasonal change of infective state of marine birnavirus in Japanese pearl oyster Pinctada fucata

Summary.  This study examines the seasonal occurrence and infective state of marine birnavirus (MABV) in cultured Japanese pearl oyster (Pinctada fucata). Planted oysters were sampled monthly in 1997 and 1998. To detect MABV in the oysters, PCR and virus isolation were carried out. Also, the indirect fluorescent antibody technique (IFAT) was performed to know the organs expressing viral antigens. The detection rate of the MABV genome by PCR was low during July to October, but increased after November. This virus was isolated only after October, with a 10–40% isolation rate. Results of the IFAT showed that the specific fluorescence was observed in hemocytes in September. Fluorescence in hemocytes decreased in January, but increased in liver parenchymal cells. These results suggest that MABV persistently infected hemocytes in summer with a small amount of genome and protein, and then the virus spread in winter into the parenchymal cells. Received November 16, 1999 Accepted May 22, 2000     

Specificity ofE. coli K-12 chromosomal segment regulating the expression of systems inhibiting flac plasmid transfer

Specificity ofE. coli K-12 chromosomal Thr-Leu-segment (genetic locus tis) regulating the expression of systems inhibiting Flac plasmid transfer is revealed. The findings point to a complex (polygenous) structure of this locus. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 559–561, November, 1997.     

Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair

A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32–37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene. Received: 27 November 1997 / 28 January 1998     

Ultrastructure of the nucleus of the Iodamoeba bütschlii cyst

The ultrastructure of the Iodamoeba bütschlii cyst from human feces was studied. The glycogen mass appears as a compact dense body in the cytoplasm without any surrounding membrane. The cytoplasm has no mitochondrion. The nucleus shows a distinct nucleolus filled with electron-dense particles. On one side of the nucleolus are electron-dense cytoplasmic masses measuring 200–400 nm. The nuclear membrane is two-layered and shows pores. Received: 27 August 1997 / Accepted: 13 November 1997     

Identification of G-protein α-subunits in ovaries

The presence of G-protein α-subunits in somatic ovarian cells of albino rats and changes in their distribution during follicle maturation are demonstrated. A possible role of these proteins in signal tranduction to ovarian structures is discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 582–585, November, 1997