The Prevalence of Listeria monocytogenes in the Environment of Dairy Farms

The prevalence of Listeria monocytogenes in the environment of dairy farms was surveyed from December 1993 to June 1994 in one city of Hokkaido. L. monocytogenes was isolated from 3 out of 5 farms investigated. Serovar 4b organism was isolated from the brain stem of a cow from one farm which was clinically diagnosed as having listeriosis. The same serovar of L. monocytogenes was also isolated from the rectal contents of a healthy cow, straw on the floor, straw in the barn, and silage scattered around the silo from the same farm. At another farm, with no reported cases of bovine listeriosis, serovar 1/2 organism was isolated from the same types of samples as the above mentioned farm except from straw on the floor. The difference in the isolation rates of the organism from straw on the floor between the two farms (22%: 5/23 vs 0%: 0/24) is considered to be caused by the different feeding methods of silage between the two farms.     

Functional Golgi units in microtubule-disrupted cultured atrial myocytes.

We examined the effects of disassembly of microtubules (MT) on the structure and the functions of the Golgi apparatus (GA) in cultured atrial myocytes. MT disassembly with nocodazole led to fragmentation of the GA into small units. The fragmented Golgi units retained their cis-trans polarity and post-cisternal elements, including the trans-Golgi network (TGN). Neither endocytosis of lectin-labeled membrane nor its delivery to the fragmented Golgi units was interrupted by fragmentation of the GA after MT disassembly with nocodazole treatment. A fraction of the secretory granules associated with the fragmented Golgi units was also labeled with the internalized tracer. These results suggest that in nocodazole-treated cultured atrial myocytes, the fragmented Golgi units appear to be structurally and functionally intact despite the altered geometric arrangement of the GA in the cells.     

Developmental arrest at early stages of Chinese hamster embryos homozygous for chromosomal rearrangements

Forty-three Chinese hamster stocks with autosomal rearrangements produced by X-irradiation were used. These rearrangements, 38 reciprocal translocations and 5 inversions, were chromosomally balanced. Heterozygotes for these rearrangements were all fertile and morphologically normal in both sexes except for one line with growth retardation. By crossing male and female heterozygotes for the same rearrangements, homozygotes were obtained in 37 lines. In the remaining 6 lines (5 with reciprocal translocations and 1 with an inversion), no homozygotes were viable. These 6 lines revealed arrested development of homozygous embryos at the two-cell stage, around the eight-cell stage, and after implantation, respectively. The bands of the breakpoints of rearrangements associated with lethality of homozygous embryos were different for each rearrangement. These results suggest that abnormal expression including embryonic lethality in homozygotes may be due to an influence of genes at the breakpoints.     

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

Summary To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph gene revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.     

Agonist-induced calcium signaling is impaired in fibroblasts overproducing inositol 1,3,4,5-tetrakisphosphate.

The proposed Ca(2+)-signaling actions of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), formed by phosphorylation of the primary Ca(2+)-mobilizing messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), were analyzed in NIH 3T3 and CCL39 fibroblasts transfected with rat brain Ins(1,4,5)P3 3-kinase. In such kinase-transfected cells, the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 during agonist stimulation was greatly increased, with a concomitant reduction in Ins(1,4,5)P3 levels and attenuation of both the cytoplasmic Ca2+ increase and the Ca2+ influx response. This reduction in Ca2+ signaling was observed during activation of receptors coupled to guanine nucleotide-binding proteins (thrombin and bradykinin), as well as with those possessing tyrosine kinase activity. Single-cell Ca2+ measurements in CCL39 cells revealed that the smaller averaged Ca2+ response of enzyme-transfected cells was due to a marked increase in the number of cells expressing small and slow Ca2+ increases, in contrast to the predominantly large and rapid Ca2+ responses of vector-transfected controls. There was no evidence that high Ins(1,3,4,5)P4 levels promote Ca2+ mobilization, Ca2+ entry, or Ca2+ sequestration. These data indicate that Ins(1,4,5)P3 is the major determinant of the agonist-induced Ca2+ signal in fibroblasts and that Ins(1,3,4,5)P4 does not appear to contribute significantly to this process. Instead, Ins(1,4,5)P3 3-kinase may serve as a negative regulator of the Ca(2+)-phosphoinositide signal transduction mechanism.     

Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs.

Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.     

Cloning and expression of two genes encoding highly homologous hemolysins from a Kanagawa phenomenon-positive Vibrio parahaemolyticus T4750 strain

We have cloned and sequenced the gene encoding thermostable direct hemolysin (TDH), a possible virulence factor in Vibrio parahaemolyticus gastroenteritis, from a Kanagawa-phenomenon-positive strain, T4750. This strain was found to contain two sequences (tdhA and tdhS) homologous to the tdh gene previously reported by Nishibuchi and Kaper [J. Bacteriol 162 (1985) 558-564] and Taniguchi et al. [Microb. Pathog. 1 (1986) 425-432]. Sequence homology of the coding regior between tdhA and tdhS was 97.2%. The deduced amino acid (aa) sequence of TdhA, excluding the putative signal peptide was identical to that of TDH protein purified from V. parahaemolyticus [Tsunasawa et al., J. Biochem. 101 (1987) 111-121] except for Glu118 instead of Gln118. Although the aa sequence deduced from the second gene, tdhS, differed in eight residues from the TDH protein, it agreed with the sequence of Tdh deduced from the previously cloned tdh gene. Both tdhA and tdhS expressed biologically active hemolysins in Escherichia coli. While the apparent molecular size of TDH purified from a culture supernatant of V. parahaemolyticus T4750 was identical to TdhA protein synthesized in E. coli, it was larger than TdhS. Only one band was detected in the culture supernatant of V. parahaemolyticus T4750 by Western blotting; its mobility was indistinguishable from that of purified TDH. These data suggest that tdhA is the structural gene for TDH found in the culture supernatant of V. parahaemolyticus T4750, and that there was only partial, if any, tdhS expression in the strain T4750 under the test conditions employed.     

Role of voltage-sensitive calcium channels in [Ca2+]i and secretory responses to activators of protein kinase C in pituitary gonadotrophs

The gonadotropin secretory response of anterior pituitary cells to phorbol esters includes both extracellular Ca2(+)-dependent and -independent components (Stojilkovi? et al, 1988; J. Biol. Chem. 263, 17301-17306, 1988). In cultured pituitary cells, measurements of [Ca2+]i using Fura-2 and of LH release during cell perifusion studies revealed that the initial effects of phorbols and permeant diacylglycerols on these responses are extracellular Ca2(+)-dependent and are mediated through activation of voltage- and dihydropyridine-sensitive calcium channels. On the other hand, pretreatment with phorbol esters for 30 to 60 min inhibited subsequent [Ca2+]i responses to diacylglycerols and phorbols and significantly reduced agonist-induced biphasic [Ca2+]i responses, with no change in the number of GnRH receptors. These findings demonstrate that protein kinase C exerts both positive and negative control of [Ca2+]i, and indicate that the calcium, phospholipid dependent enzyme participates in the activation of voltage-sensitive calcium channels and hormone secretion in pituitary gonadotrophs.     

Splicing defect of the glycoasparaginase gene in two Japanese siblings with apartylglucosamimiria

Summary Molecular analysis of the glycoasparaginase gene was performed on two Japanese siblings with aspartylglucosaminuria. The cDNA from one patient contained 7 additional bases between exons 3 and 4 (3-terminal sequence of intron 3). This insertion resulted in a frame shift, and a termination codon appeared at amino acid 146. Amplification and sequencing of genomic DNA detected a single base transition (AG) at the 5 side adjacent to the insertion sequence. This mutation created a consensus AG dinucleotide in the splice acceptor site, and produced almost exclusively an abnormal mRNA containing the insertion by alternative splicing. The calculation of the sample score of the acceptor site supported this analytical result. BsmAI restriction site analysis of amplified cDNA and genomic DNA showed that these patients were homozygotes for this mutation. We conclude that the splicing defect in intron 3 causes glycoasparaginase deficiency in these patients.