Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity     

A genetic study of the human low-voltage electroencephalogram

Summary The studied phenotype, the low-voltage electroencephalogram (LVEEG), is characterized by the absence of an alpha rhythm from the resting EEG. In previous studies, evidence was found for a simple autosomal-dominant mode of inheritance of the LVEEG. Such a polymorphism in brain function can be used as a research model for the stepwise elucidation of the molecular mechanism involved in those aspects of neuronal activity that are reflected in the EEG. Linkage with the variable number of tandem repeats (VNTR) marker CMM6 (D20S19) and localization of an LVEEG (EEGV1) gene on 20q have previously been reported, and genetic heterogeneity has been demonstrated. This latter result has been corroborated by studing new marker (MS214). The phenotype of the LVEEG is described here in greater detail. Its main characteristic is the absence of rhythmic alpha activity, especially in occipital leads, whereas other wave forms such as beta or theta waves may be present. Analysis of 17 new families (some of them large), together with 60 previously described nuclear families, supports the genetic hypothesis of an autosomal-dominant mode of inheritance. Problems connected with the analysis of linkage heterogeneity, exclusion mapping, and the study of multipoint linkage are discussed. A possible explanation of the localization of LVEEG in the close vicinity of another gene influencing synchronization of the normal EEG, the gene for benign neonatal epilepsie, is given.     

Embryonic development and incidence of aneuploidy in two rabbit strains of different fecundity

The correlation between strain fecundity and (i) development and (ii) rate of aneuploidy was studied in rabbit preimplantation embryos obtained from 2 strains of different fecundity. Embryos were investigated at Days 3-6 (preimplantation development) or Days 2, 4 and 6 post coitum (aneuploidy). Embryonic size and cell proliferation varied on the days of investigation, but with no consistent tendency in favour of one strain. The incidence of aneuploidy did not differ significantly between embryos from the 2 strains (P greater than 0.05). The multifactorially determined criterion of prolificacy was not selectively correlated with overall differences in embryonic preimplantation growth and rate of aneuploidy.     

The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases.

A novel method has been developed to allow cloning of protein targets for receptors with tyrosine kinase activity. By utilizing the carboxy-terminal tail of EGF receptor (EGFR) as a probe to screen lambda gt11 expression libraries, several EGFR-binding proteins have been cloned; two have been analyzed and contain unique SH2 and SH3 domains. One gene (GRB-1) has been fully sequenced, is expressed in various tissues and cell lines, and has a molecular mass of 85 kd. Interestingly, GRB-1 encodes the human counterpart of the PI3 kinase-associated protein p85. Advantages of this technique include the ease of cloning tyrosine kinase receptor targets present at low levels and the ability to identify proteins that are related in their capacity to bind activated receptors but contain no significant DNA sequence homology. This method, termed CORT (for cloning of receptor targets), offers a general approach for the identification and cloning of various receptor targets.     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida,Polychaeta)

Summary Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to: A.W.C. Dorresteijn     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.