Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis.

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Microbiological Assay for Organic Compounds in Seawater. II: Distribution of Adenine, Uracil, and Threonine

Biochemically deficient strains of Serratia marinorubra have been isolated with specific requirements for adenine, uracil, and threonine. Standard curves for dose to growth response have been obtained showing a linear sensitivity from 0.5 to 4.0 mg of adenine per liter of seawater, 0.1 to 2.0 mg of uracil per liter of seawater, and 0.5 to 10 mg of threonine per liter of seawater. These mutants have been used to test for the presence of their required metabolites in natural seawater samples from the Gulf of Mexico and adjacent bays. Of the three compounds under investigation, adenine was found in 10 samples, uracil in 2 samples, and threonine in none. The possible significance of these findings to the marine environment is discussed.     

Histoplasmosis and Thrombocytopenic Purpura: Transmission by Renal Homotransplantation

Two patients with disseminated histoplasmosis are reported. One patient presented with severe thrombocytopenic purpura and splenomegaly. Histoplasmin skin test, blood and bone marrow cultures and smears, sputum cultures, and chest radiographs were negative for Histoplasma capsulatum. She died on the sixth hospital day from a massive intracerebral hemorrhage. Cardiorespiratory function was maintained until one kidney was removed for homotransplantation. The second patient, with chronic glomerulonephritis and uremia, received the renal homograft from the first patient. Initial signs of homograft rejection developed five days postoperatively. Diffuse thrombocytopenic purpura occurred shortly thereafter. Spores of Histoplasma capsulatum were observed in blood smears, in leukocyte concentrates, and in five-day leukocyte cultures from the blood obtained prior to death. Disseminated histoplasmosis was found in both patients at autopsy. The severe platelet deficit in both cases suggests that systemic histoplasmosis should be considered as a cause of thrombocytopenic purpura.To our knowledge, this is the first reported instance of direct transmission of Histoplasma capsulatum, and must be considered a hazard in homotransplantation.In vitro leukocyte cultures as a method for early diagnosis of certain disseminated fungous infections needs further investigation.     

Influence of SQ 30741 on thromboxane receptor-mediated responses in the feline pulmonary vascular bed.

The effects of SQ 30741, a thromboxane A2 (TxA2) receptor blocking agent, on responses to the TxA2 mimic, U-46619, were investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. The administration of SQ 30741 in doses of 1-2 mg/kg iv markedly reduced vasoconstrictor responses to U-46619 without altering responses to prostaglandin (PG) F2 alpha or PGD2 and serotonin. SQ 30741 had no significant effect on mean vascular pressures in the cat, and the dose-response curve for U-46619 was shifted to the right in a parallel manner with a similar apparent maximal response. In addition to not altering responses to PGF2 alpha, PGD2 alpha, or serotonin, SQ 30741 (2 mg/kg iv) was without significant effect on pulmonary vasoconstrictor responses to the PGD2 metabolite 9 alpha, 11 beta-PGF2, norepinephrine, angiotensin II, BAY K 8644, endothelin 1, or endothelin 2. Although responses to vasoconstrictor agents, which act through a variety of mechanisms, were not altered, responses to the PG and TxA2 precursor, arachidonic acid, were reduced significantly. The duration of the TxA2 receptor blockade was approximately 30 and 75 min at the 1- and 2-mg/kg iv doses of the antagonist, respectively. The present data show that SQ 30741 selectively blocks TxA2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed. These data suggest that responses to arachidonic acid are due in large part to the formation of TxA2 and that discrete TxA2 receptors unrelated to receptors activated by PGD2 or PGF2 alpha are most likely located in resistance vessel elements in the feline pulmonary vascular bed.     

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.     

DNA sequence of a class I pseudogene from theTla region of the murine MHC: Recombination at a B2 Alu repetitive sequence

Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1^c gene from theTla ^c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1^c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1^c gene is structurally related to the previously sequenced T13^c gene that encodes a serologically defined TL antigen. The 3 half of the T1^c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13^c gene; however, the 5 half of the T1^c gene has little homology with the T13^c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13^c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.     

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.     

Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products

 The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual’s immune response, and may also contribute to susceptibility to diseases such as autoimmunity. Received: 9 January 1996 / Revised: 22 February 1996