Asynchrony between pronuclear development and protein synthetic changes in zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo

The pattern of proteins synthesized by one-cell embryos derived from unaged oocytes and oocytes aged postovulation in vivo was analyzed by means of 35S-methionine labeling and gel electrophoresis. The oocytes were obtained after ovulation induction by an injection of luteinizing hormone-releasing hormone (LHRH) at proestrus or after a superovulation procedure. The analysis was performed in unfertilized aged and unaged secondary oocytes and in zygotes derived from them. The patterns of proteins synthesized by secondary oocytes from all experimental groups were very similar: The oocytes showed a predominant synthesis of 35 kDa proteins. Zygotes from aged as well as unaged LHRH-induced oocytes also showed a predominant synthesis of one group of polypeptides with a relative molecular weight of about 35 kDa. The proteins of the 35 kDa protein complex migrated in an upper (u), middle (m), or lower (l) band in 10% polyacrylamide sodium dodecyl sulfate (SDS) gels. The u- and m-band 35 kDa proteins were shown to be synthesized by secondary oocytes. Early pronuclear zygotes from unaged LHRH-induced oocytes synthesized u- and m- but no l-band 35 kDa proteins. In contrast, part (38%, n = 47) of the early pronuclear zygotes from aged LHRH-induced oocytes did synthesize the l-band 35 kDa proteins. Late pronuclear zygotes (LPZ) from aged as well as unaged oocytes synthesized predominantly the l-band 35 kDa proteins. However, although only 5.8% (n = 51) of LPZ from unaged oocytes synthesize m- and l-band 35 kDa proteins, these bands of proteins are present in 25% (n = 24) of the LPZ from aged oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer membrane proteins

Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.     

The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology

Background

Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers.

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Analysis of Streptococcal Cell Wall Fractions by Curie-Point Pyrolysis Gas-Liquid Chromatography

A streptococcal strain, classified as Z(3)III was differentiated from its mutant strain, Z(3), lacking the type III polysaccharide antigen, by Curie-point pyrolysis gas-liquid chromatography. Differences observed in pyrograms of whole cells or cell envelopes of both strains could be directly related to the pyrolysis pattern of the purified type III antigen. The same results were obtained when streptococcus F III and its mutant were analyzed. Whereas the pyrolysis patterns of the type III antigen extracted from Z(3)III and F III bacteria were identical, marked differences were found in pyrograms of the serologically identical type III antigen isolated from the culture medium. Type III antigen was also easily differentiated from the purified type I, II and IV antigens. From the above findings it was concluded that pyrolysis gas-liquid chromatography can be used as a tool for the quality control and identification of streptococcal cell wall components.     

The type antigen III of group F streptococci separation of group and type antigens and partial characterization of type III antigen

Polysaccharides extracted from Z₃III streptococci either with formamide or with dilute hydrochloric acid or isolated from the growth medium could be fractionated in type III- and group Z₃ antigens by alcohol precipitation. No good separation could be obtained from TCA extracts. When the same extractions and fractionations were applied to streptococci carrying type III antigen, but different group antigens, good separations were again obtained of all formamide extracts, but not of all hydrochloric acid extracts. The group antigens showed a rhamnose content of at least 50% and contained hexosamines. Type III antigens contained mainly rhamnose, glucose and galactose in relative amounts of approximately 1:2:3. Analysis of the methylated type III antigen suggests it to be a polysaccharide with a linear structure. Type III antigen isolated from the medium was characterized not only by a different sugar ratio, but also by its fucose content of 20%. In some cases the purified polysaccharides contained considerable amounts of glycogen-like material. Partial acid hydrolysis of the type III antigen extracted with formamide yielded a great number of oligosaccharides. Analyses, inhibition reactions and methylation studies gave indications that the most probable structure of a determinant group of type III antigen is β-glucosyl-(1-6)-galactosyl-(1-6)-galactosyl-(1-3)-rhamnose. The possibility of the existence of a second determinant group is discussed.