Zinc, a novel structural element found in the family of bacterial adenylate kinases.

The adk gene from Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli under the control of the lac promoter. The primary structure of B. stearothermophilus adenylate kinase exhibited 76% identity with the enzyme from Bacillus subtilis, 60% identity with the enzyme from Lactococcus lactis, and 42% identity with the enzyme from E. coli. The most striking property of the adenylate kinase from B. stearothermophilus is the presence of a structural zinc atom bound to four cysteines in a zinc finger-like fashion. The ability to coordinate zinc is predicted also for a number of other isoforms of bacterial adenylate kinases. Furthermore, the tightly bound metal ion contributes to the high thermodynamic stability of adenylate kinase from B. stearothermophilus.     

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     

Unusual properties of genomic DNA molecules spanning the euchromatic-heterochromatic junction of a Drosophila minichromosome.

While investigating the copy number of minichromosome Dp(1;f)1187 sequences in the polyploid chromosomes of ovarian nurse and follicle cells of Drosophila melanogaster we discovered that restriction fragments spanning the euchromatic-heterochromatic junction of the chromosome and extending into peri-centromeric sequences had the unusual property of being selectively resistant to transfer out of agarose gels during Southern blotting, leading to systematic reductions in Dp1187-specific hybridization signals. This property originated from the peri-centromeric sequences contained on the junction fragments and was persistently associated with Dp1187 DNA, despite attempts to ameliorate the effect by altering experimental protocols. Transfer inhibition was unlikely to be caused by an inherent physical property of repetitive DNA sequences since, in contrast to genomic DNA, cloned restriction fragments spanning the euchromatic-heterochromatic junction and containing repetitive sequences transferred normally. Finally, the degree of inhibition could be suppressed by the addition of a Y chromosome to the genotype. On the basis of these observations and the fact that peri-centromeric regions of most eukaryotic chromosomes are associated with cytologically and genetically defined heterochromatin, we propose that peri-centromeric sequences of Dp1187 that are incorporated into heterochromatin in vivo retain some component of heterochromatic structure during DNA isolation, perhaps a tightly bound protein or DNA modification, which subsequently causes the unorthodox properties observed in vitro.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

A requirement for three cell types for in vitro generation of specific secondary cell-mediated cytotoxic response against SV40-induced tumor-associated antigens in mice

The present study demonstrates that a collaborative interaction among three cell types, namely, two distinct subsets of T cells and macrophages is needed for in vitro generation of specific secondary cell-mediated cytotoxicity against syngeneic SV40-transformed cells in mice. These data suggest a central role of three cell types in the generation of efficient antitumor immune response in syngeneic tumor systems.     

Distinct distributions of D-erythro-neopterin in arteries and veins and its recovery by an enterohepatic circulation.

Large amounts of D-erythro-neopterin, a pteridine derivative, are formed from guanosine triphosphate (GTP) by human macrophages upon stimulation with interferon-gamma. In addition, in humans a basal neopterin level in all body fluids is evident also in absence of immunological stimuli. Extremely high concentrations of D-erythro-neopterin were detected in biliary fluid. We therefore investigated, if an enterohepatic circulation might exist for this substance. We quantified concentrations of pteridines in serum obtained from various vessels and in biliary fluid. Samples were collected during surgery of five patients with duodenal ulcer or adenocarcinoma of the stomach. Our data clearly demonstrate the existence of an enterohepatic circulation for the recovery of neopterin which seems to be specific for this substance. The relative distributions of neopterin concentrations in the gastrointestinal tract and vessels were seen invariably in all patients and were consistent with findings in five corpses examined post mortem. In addition, significantly higher neopterin concentrations, were found in arteries than in veins. The data indicate that neopterin derivatives are consumed in the peripheral capillary system and an enterohepatic circulation is established to maintain constant blood levels of neopterin derivatives. Furthermore, we suppose that the liver is the source of constitutive neopterin concentrations.     

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.     

Chondroitin SO4 catabolism in chick embryo chondrocytes

An enzyme preparation from cultured chick embryo vertebral chondrocytes attacks chondroitin SO4 oligosaccharides from the nonreducing terminal in a recycling pathway involving the sequential action of a beta-glucuronidase, a 4- or a 6-sulfatase, and a beta-N-acetylgalactosaminidase. The sequence is blocked by saccharo-1,4-lactone, an inhibitor of the beta-glucuronidase, or by 2-acetamido-2-deoxy-D-galactonolactone, an inhibitor of the beta-N-acetylgalactosaminidase. The level of 4-sulfatase activity is low relative to the other activities and limits the rate of catabolism of hybrid oligosaccharide structures containing both 6-sulfated galactosamine residues and 4-sulfated galactosamine residues. This results in the accumulation of shortened oligosaccharides, most of which have galactosamine-4-SO4 residues at their nonreducing terminals. In the presence of the lactone inhibitors, polymeric chondroitin SO4 is broken down by the enzyme preparation to oligosaccharides which are 10 to 15 monosaccharides long, indicating that degradation of chondroitin SO4 chains is initiated by an endoglycosidase which generates oligosaccharide substrates for the recycling exoglycosidase system.