Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase

An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.     

A new mitochondrial disease associated with mitochondrial DNA heteroplasmy.

A variable combination of developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy occurred in four members of a family and was maternally transmitted. There was no histochemical evidence of mitochondrial myopathy. Blood and muscle from the patients contained two populations of mitochondrial DNA, one of which had a previously unreported restriction site for AvaI. Sequence analysis showed that this was due to a point mutation at nucleotide 8993, resulting in an amino acid change from a highly conserved leucine to arginine in subunit 6 of mitochondrial H(+)-ATPase. There was some correlation between clinical severity and the amount of mutant mitochondrial DNA in the patients; this was present in only small quantities in the blood of healthy elderly relatives in the same maternal line.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F₁ of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

Elimination of multiple innervation in the developing avian superior oblique muscle (1)

Motor endplates in the developing avian superior oblique muscle first appear on day 18 of incubation. Most of the endplates from this time through hatching (day 27) are innervated by multiple fibers. Each endplate in the post-hatching period is innervated by only one fiber. Time of elimination of multineuronal innervation does not correlate with the time of trochlear neuron loss; the former occurs much later in development. Removal of multiple innervation is therefore, not the cause of the naturally occurring neuron loss.     

Capnocytophaga: New genus of Gram-negative gliding bacteria. II. Morphology and ultrastructure

Gram-negative, anaerobic gliding bacteria were isolated from normal supragingival plaque and from periodontal lesions. Isolates could be divided into two size classes: small 2.4–4.2 m×0.38–0.5 m and large 4.8–5.8 m×0.42–0.6 m cells. The outer membrane was either loose-fitting and wavy, or taut, and of variable thickness. An electron-dense fuzz was discernible on several of the isolates. The periplasmic region was of variable electron-density. The genus Capnocytophaga has been proposed for these organisms based on morphological and cultural characteristics.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.