DNA fingerprinting of lactococci and streptococci used in dairy fermentations

Summary A DNA fingerprinting procedure was developed for strains of Lactococcus lactis subsps. lactis and cremoris, biovar. diacetylactis, and Streptococcus salivarius subsp. thermophilus, used in dairy fermentations. Total cellular DNA was extracted and digested with restriction endonucleases, HindIII or HaeIII, followed by separation of the fragments using agarose gel electrophoresis. L. lactis C2 was used as a representative strain for examining the effect of growth phase and cell concentration, cell washing conditions prior to lysis, type and concentration of the enzyme used to digest the cell wall, composition of the lysis buffer, and gel electrophoresis conditions. Following optimization of the fingerprinting procedure, electrophoretic migration of fragments from 23 strains produced reproducible gel patterns. L. lactis subsp. lactis strains ML3 and C2 appeared to be identical when restrricted with either Hind III or HaeIII. Similarly, S. salivarius subsp. thermophilus strains 19987 and 19258, and L. lactis subsp. cremoris strains 134 and C3, appeared to have identical DNA fingerprints following digestion with HindIII. To determine the usefulness of this technique for monitoring population changes during fermentation, various ratios of two closely related strains were inoculated into milk and allowed to grow for 16 h at 32° C. The initial inoculum ratios were determined by standard plate counts, and the final ratio was deterimined by DNA fingerprinting. DNA fingerprinting will be useful in the identification, characterization, and comparison of food fermentation microorganisms.Published as paper No. 17,803 of the contribution series of the Minnesota Agricultural Experiment Station Offprint requests to: S. K. Harlander     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

On the origin of lateral asymmetry

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.     

Dust and ammonia in horse stalls with different ventilation rates and bedding

Airborne particles and ammonia were monitored in horse stalls managed under four conditions. Two ventilation rates, high (27 air changes per h) and low (5 air changes per h) and two bedding types, paper and straw, were employed. At both ventilation rates, the number of airborne particles generated while the stalls were mucked out was higher with straw than with paper. Particles were more efficiently cleared at the higher ventilation rate in both the straw and paper stalls. Ammonia measurements reflected an accumulation over time. In the stalls with low ventilation, ammonia levels were significantly higher than in those stalls with high ventilation regardless of bedding type. Management decisions and their relationships to respiratory disorders are discussed.     

Taxonomic and biochronological significance of specimens of the Triassic dicynodontDinodontosaurus Romer 1943 in the Tübingen collection

Two species of the dicynodontDinodontosaurus from the Middle Triassic interval of the Santa Maria Formation in Rio Grande do Sul, southern Brazil,D. tener (von Huene 1935) andD. turpior (von Huene 1935), are based on undiagnostic lectotypes and thus arenomina dubia. The oldest valid, available name for a species ofDinodontosaurus isD. oliveirai Romer 1943, the type species of the genus. (The unused senior subjective synonymDiodontosaurus pedroanum Caldas, 1936 has been suppressed by the International Commission on Zoological Nomenclature).Chanaria platyceps Cox 1968 andDinodontosaurus brevirostris Cox 1968 are junior subjective synonyms ofD. oliveirai Romer 1943.Dinodontosaurus thus is a monospecific genus known from the Santa Maria Formation and the Ischichuca (= Chañares) Formation of northwestern Argentina. This dicynodont and associated tetrapods characterize the Chanarian land-vertebrate faunachron, which is of Middle Triassic age, probably Ladinian.     

Sabal palmetto seed size: causes of variation,choices of predators,and consequences for seedlings

High variation in seed size, as is common among angiosperms, may be maintained in a plant species when several factors select for seed size. Variation may also result from differences among adult plants, such as nutrient and water availability or the amount of photosynthetic tissue. In a study of Sabal palmetto seed ecology I found high seed size variation both within- and among-palms, and investigated possible factors maintaining this variation. Seed size was positively correlated with the number of leaves on parent palms. Larger seeds produced more vigorous seedlings that had greater leaf length, area, and mass, and greater root mass. Caryobruchus gleditsiae (Bruchidae: Coleoptera), whose larvae develop within palm seeds, preferentially oviposited on larger seeds, which in turn produced larger beetle offspring. By choosing the largest seeds available, ovipositing beetles thus affect both the quantity and the quality of seeds available for recruitment. I conclude that because beetle predation selects against large seeds, while larger seeds promote seedling vigor, the maintenance of seed size variation may be an adaptation of S. palmetto promoting both seed escape from predators and seedling vigor.     

A new major histocompatibility complex class I b gene expressed in the mouse blastocyst and placenta

 Because of the role major histocompatibility complex (MHC) class I b molecules may play during mouse embryonic development, we thought it would be interesting to search for additional MHC class I b molecules that might be expressed in preimplantation embryos, and in particular in the trophoblastic lineage. We therefore screened a mouse preimplantation blastocyst cDNA library for MHC class I sequences. This search led to the identification and characterization of a new MHC class I b gene, blastocyst MHC. Sequences identical to the exons and 3′ untranslated region of this gene have been found in many laboratory mouse strains, as well as in the related mouse species Mus spreciligus. The presence of this gene in mouse strains of different MHC class I haplotypes argues that blastocyst MHC is a unique, newly-described gene rather than a new allele of a previously described mouse MHC class I gene. Blastocyst MHC has the structure of an MHC class I b gene, with the six exons characteristic of T-region genes. It is linked to H2-D. The amino acid sequence encoded by this gene maintains all the features of a functional antigen-presentation domain. The blastocyst MHC gene, like the human class I b gene HLA-G, is expressed at the blastocyst stage and in the placenta, and may be the mouse analog for HLA-G. Received: 31 May 1996 / Revised: 19 August 1996     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.