A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Benthic bacterial biomass supported by streamwater dissolved organic matter

Bacterial biomass in surface sediments of a headwater stream was measured as a function of dissolved organic carbon (DOC) flux and temperature. Bacterial biomass was estimated using epifluorescence microscopic counts (EMC) and ATP determinations during exposure to streamwater containing 1,788g DOC/liter and after transfer to groundwater containing 693g DOC/liter. Numbers of bacteria and ATP concentrations averaged 1.36×10⁹ cells and 1,064 ng per gram dry sediment, respectively, under initial DOC exposure. After transfer to low DOC water, biomass estimates dropped by 53 and 55% from EMC and ATP, respectively. The decline to a new steady state occurred within 4 days from ATP assays and within 11 days from EMC measures. A 4°C difference during these exposures had little effect on generation times. The experiment indicated that 27.59 mg/hour of natural DOC supported a steady state bacterial biomass of approximately 10g C/g dry weight of sediment (from EMC determinations). Steady state bacterial biomass estimates on sediments that were previously muffled to remove organic matter were approximately 20-fold lower. The ratio of GTPATP indicated differences in physiological condition or community composition between natural and muffled sediments.     

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.     

AT-1.1: A thymus-specific alloantigen of chickens

A thymocyte-specific alloantigen, designated AT (avian thymus) –1.1, has been detected in Cornell C strain (CS) and Obese strain (OS) chickens, the latter being a strain derived from CS which develops a spontaneous form of autoimmune thyroiditis (SAT). Antisera specific for this antigen were developed first in a turkey immunized with thymocytes from an OS chicken and, later, in AT-1.1-negative CS chickens immunized with AT-1.1-positive thymocytes. AT-1.1 was detected in 50–70% of cells in a thymus cell suspension, but was not seen on peripheral blood lymphocytes, erythrocytes, or cells from bursa, spleen, kidney, liver, or brain. It was present on thymocytes of chickens at all ages tested, from 1 day to 6 months of age. AT-1.1 was not detected in six chicken lymphoid tumor cell lines tested, and birds expressing it were found to be negative for the presence of Marek's disease viral antigens. Pedigree studies on 287 (OS × CS)F₂ chickens demonstrated that AT-1.1 is expressed in a dominant or codominant manner, and the gene coding for this antigen was not linked to the B (major histocompatibility) complex. The genetics and tissue distributions of AT-1.1 indicate that it differs from thymus cell surface antigens, avian or mammalian, previously described.     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Preliminary results from a controlled evaluation of thermal biofeedback as a treatment for essential hypertension

In a controlled trial, thermal biofeedback (n=20) and abbreviated progressive relaxation (n=22) were compared in the treatment of mild to moderate hypertensive patients whose blood pressures (BP) were initially controlled on two medications. For the clinical end point of maintaining control of BP on a single drug after treatment, biofeedback was superior to relaxation training (at 3 months, 47% success for biofeedback versus 23% for relaxation). This same result tended to be true for patient-measured home BPs. BPs from laboratory psychophysiological testing showed no consistent advantage for one treatment over the other.This research was supported by a grant from NHLB1, HL-27622.     

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bateria

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type — a, b and c — in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250–1700 cm^?1, were obtained with tunable dye laser excitation in the wavelength range 550–600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.     

Molecular conformations and 8-CH exchange rates of purine ribo- and deoxyribonucleotides: investigation by Raman spectroscopy

The rate of deuterium exchange of the 8-CH group in a purine deoxyribonucleotide, is the same as the 8-CH exchange rate in the corresponding purine ribonucleotide, with the exception of 5′-nucleotides of guanine. The observed 20% slower rate of 8-CH exchange in 5′-dGMP versus 5′-rGMP, over the temperature range 50–80°C, are attributable to differences in molecular conformation, including differences in ring puckering of the furanose substituents. Minor differences in 8-CH exhange rates are observed between 5′-and cyclic (3′:5′)-deoxyribonucleotides of a given purine, which are similar to those observed previously between corresponding 5′- and cyclic ribonucleotides that have been attributed to the charge difference of their respective phosphate groups [Ferreira, S. A. & Thomas, G. J., Jr. (1981) J. Raman Spectrosc. 11 , 508–514]. The coupling of guanine and furanose ring structures in the 5′-nucleotides is also evident from the vibrational frequencies of the guanine ring, which are strongly dependent on the pucker of the attached furanose moiety. Raman difference spectroscopy clearly reveals the dependence of purine nucleotide spectra on sugar-ring pucker. In the case of GMP, the guanine characteristic ring breathing mode near 600–700 cm^?1 depends for its exact position and intensity on the proportion of C3′-endo (668 cm^?1) and C2′-endo (682 cm^?1) conformers in equilibrium with one another. The Raman intensity ratio I(668)/I(682) is proposed as a measure of the conformer ratio C3′-endo/C2′-endo in 5′-dGMP with possible application also to nucleic acids. Among cyclic nucleotides, differences in spectra of deoxyribo- and ribo- forms also appear to be related to differences of molecular conformation.     

Bacterial Symbionts in the Sugar Beet Root Maggot, Tetanops myopaeformis (von Röder)

Aerobic heterotrophic and facultative anaerobic bacteria were isolated from all developmental stages of the sugar beet root maggot, Tetanops myopaeformis (von Röder). Two distinct bacterial symbiotic relationships were observed. Serratia liquefaciens and Serratia marcescens were found to be associated with all developmental stages. Bacterial symbiont transmission occurred from one generation to the next. Symbionts were transferred from the male reproductive system to the female reproductive system, where both an internal infiltration of the egg chorion and an external smearing of the eggs occurred during oviposition. Pseudomonas maltophilia was found in association with the larval gut and the inner surface of the puparium. Electron microscopy of the inner puparial surface revealed symbionts within the chitinous wall. In vitro symbiont chitinase production was found, using both nephelometric (turbidimetric) and N-acetylglucosamine assays. A relationship appeared to exist between adult fly emergence and enzymatic chitin degradation of the puparium by the bacterial symbionts.