Factors contributing to the seasonal variation of Bacillus spp. in pasteurized dairy products

The seasonal variation in the spoilage of pasteurized products, especially double cream, by spore-forming bacteria was due to a number of factors. By far the most important was the seasonal variation in the types of organisms isolated from raw milks. Psychrotrophic spore-formers predominated in the summer-autumn months and these strains were able to germinate rapidly and grow in refrigerated dairy products. There was evidence that the concentration of one or more factors which promoted germination of psychrotrophic strains of Bacillus spp. in milk was higher during the summer than in the winter. This again may contribute to seasonal differences in spoilage by spore-forming bacteria. Post-heat treatment contamination by spores of Bacillus spp. may also be more prevalent in the summer-autumn period and evidence was obtained that spores associated with post-pasteurization contamination could germinate and grow more rapidly than those introduced into the product from the raw material. Thus, the increased spoilage of pasteurized products by Bacillus spp. observed in the June to October period may be due to a combination of factors. The relative contribution that each makes is not easily resolved.     

Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.     

Chromosome banding in salmonid fish: nucleolar organizer regions in Salmo and Salvelinus

Chromosome banding patterns obtained by silver staining (Ag-NORs) were analyzed in three species of Salmo (rainbow, brown trout, and Atlantic salmon) and three species of Salvelinus (brook trout, lake trout, and arctic char). In rainbow trout and Atlantic salmon the Ag-NORs were found at the secondary constrictions of a single chromosome pair, while in brown trout the Ag-NORs were found on the short arms of one or two of the two longest subtelocentric or acrocentric chromosome pairs. The location of the Ag-NORs was multichromosomal in the three Salvelinus species, occurring on one or both members of four to six different chromosome pairs in different individuals. The Ag-NOR sites were on the short arms of some acrocentric pairs and at the telomeres of other acrocentric pairs and one or two metacentric pairs. Chromomycin A3 positive bands were found at the same sites as the Ag-NORs in all species. In the species with multichromosomal location of Ag-NORs, polymorphisms in the size and location of the NORs were extremely common, so that almost every individual fish had a different pattern of Ag-NOR sites.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenstr?m IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.     

Mammalian fertilization as seen with the scanning electron microscope

For several years we have been looking at mammalian gametes and their interactions with the scanning electron microscope (SEM). Examining the images produced by the SEM has given us a three-dimensional view of sperm, eggs, and egg investments. We are particularly impressed with the structural variation among gametes of different mammalian species. In this short report we examine the structure of mammalian spermatozoa, eggs, zonae pellucidae, and cumuli. Our observations and those of others have led us to believe that variation in gamete structure and function may have evolved as a mechanism for reproductive isolation of mammalian species.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.