HLA patterns in pernicious anaemia.

The pattern of HLA antigens was studied in 127 patients with Addisonian pernicious anaemia. The pattern in the whole group of patients differed significantly from that in 586 controls. But different subgroups of the patients had different HLA antigens. Among 27 patients with anaemia associated with endocrine disease there was an increased frequency of HLA-B8, B18, and BW15. The remaining 100 patients, who did not have endocrine disease, showed increased frequencies of HLA-B7 and B12. The positive association with HLA-B12 among this subgroup was confined to 62 patients with severly impaired vitamin B12 absorption, including 13 patients with vitamin B12 neuromyelopathy, who had the highest frequencies of HLA-B7 and B12. The significant heterogeneity in HLA patterns in different clinical subgroups of these patients indicates genetic heterogeneity in pernicious anaemia and explains previous discrepancies in the associations between HLA antigens and pernicious anaemia.     

The effect of prostaglandin E2 on the cardiovascular response to angiotensin II in pregnant rabbits

The rise in arterial blood pressure in response to angiotensin II was studied in the last third of pregnancy in rabbits. The response was compared with that of pregnant rabbits during infusion of prostaglandin E₂ and F_2α. Prostaglandin E₂ significantly diminished the rise in diastolic pressure in response to angiotensin II. Prostaglandin F_2α did not alter the response. Intravenous indomethacin elevated the blood pressure and caused an absolute increase in the pressor response. It did not mediate a change in the percentage rise in blood pressure in response to angiotensin II.     

Tamoxifen fails to block estradiol accumulation, yet is weakly accumulated by the juvenile zebra finch anterior hypothalamus: an autoradiographic study.

In experiment 1, we used autoradiographic procedures to examine whether tamoxifen could displace 3H-estradiol labeling in the anterior hypothalamus and the caudal nucleus of the ventral hyperstriatum (HVc) of ovariectomized 20-day-old female zebra finches. There was no significant reduction in labeling of cells by 3H-estradiol in birds preinjected with unlabeled tamoxifen. In experiment 2, we found that injections of 3H-tamoxifen caused-weak labeling of cells in the anterior hypothalamus of 20-day-old male and female zebra finches. These results are compatible with the idea that tamoxifen does not block the action of estradiol in the brain of zebra finches, and suggest that the effects of early tamoxifen treatment on the morphology of the song system may reflect central actions of tamoxifen.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion [Lederer, F., & Mathews, F.S. (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin]. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Virus-associated RNAs of naturally occurring strains and variants of group C adenoviruses

We compared the sequences of the virus-associated (VA) RNAs of group C adenoviruses, serotypes 1, 2, 5, and 6, and of three variants of adenovirus type 2 (Ad2) selected for loss of the BamHI restriction site in the VA RNAI gene. In the naturally occurring strains. VA RNAI exists in two forms which differ by two nucleotides: one form is found in Ad2 and Ad6, and the other is found in Ad1 and Ad5. There are three sites of variation in Va RNAII, the Ad1, Ad2, and Ad5 forms each differing from Ad6 VA RNAII at one of the positions. One of the selected variants has a four-base duplication within the BamHI cleavage site, whereas the two others have acquired a VA RNAI sequence indistinguishable from that of Ad5. The findings are interpreted in terms of the secondary structures of the VA RNAs and the interrelationships among the viruses.     

PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.