Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献   

Plasma norepinephrine concentrations: No differences among normal volunteers and low,high or normal renin hypertensive patients

Plasma norepinephrine concentrations were measured by a sensitive radioenzymatic method in 51 patients with essential hypertension and 26 age-matched normal volunteers under conditions of ad libitum sodium intake, after volume expansion by infusion of saline intravenously, and after volume contraction by administration of furosemide orally. The hypertensive patients were classified into low, normal and high renin groups both by renin-sodium indexing and by their renin response to furosemide and saline administration. Plasma norepinephrine concentrations were similar among normal volunteers and patients with low, normal or high renin hypertension while the people were either recumbent or after they stood for 5 min. These and other results do not support the hypothesis that abnormal activity of the sympathetic nervous system accounts for the low or high renin values seen in many hypertensive patients.     

Dynamical similarity and the theory of biological transformations

The D'Arcy Thompson concept of biological transformations is developed in a form analogous to such physical concepts as the Law of Corresponding States in thermodynamics, and the Principles of Similitude found in engineering. We find that such concepts depend on a distinction between fundamental and derived quantities, in which the values assigned to the fundamental quantities set the natural scales for the derived ones. Among other things, we see that critical phenomena, such as phase transitions, arise as an immediate consequence of this distinction. In a biological context, we explore the implications of Thompson's hypothesis that closely related organisms are phenotypically similar, assuming that the organisms we see are the result of selection processes operating on phenotypes.     

Analysis of DNAs from two species of the virilis group of Drosophila and implications for satellite DNA evolution

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.     

Degradation of HeLa S3 cell chromatin by auromomycin and its chromophore

The antitumor protein agent auromomycin was found to degrade chromatin structure primarily by inducing strand scissions in linker regions. The reaction was stimulated by dithiothreitol. The chromophore form of the drug caused similar effects on chromatin, but it appeared to function at a more rapid rate. There was no evidence that auromomycin could cause breakage in core regions of chromatin.     

Amino-terminal sequences of the L, M, and H subunits of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

We have determined the sequence of the 25-28 amino-terminal residues of the three subunits, L, M, and H, of the membrane-bound reaction center protein of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. The sequences are as follows: L, H2N-Ala-Leu-Leu-Ser-Phe-Glu-Arg-Lys-Tyr-Arg- Val-Pro-Gly-Gly-Thr-Leu-Val-Gly-Gly-Asn-Leu-Phe-Asp-Phe-(His)-Val-; M, H2N-Ala-Glu-Tyr-Gln-Asn-Ile-Phe-Ser-Gln-Val-Gln-Val-Arg-Gly-Pro-Ala-Asp-Leu-Gly-Met-Thr-Glu-Asp-Val-Asn-Leu-Ala-Asn-; H, H2N-Met-Val-Gly-Val-Thr-Ala-Phe-Gly-Asn-Phe-Asp-Leu-Ala-Ser-Leu-Ala-Ile-Tyr-Ser-Phe-Trp-Ile-Phe-Leu-Ala-X-Leu-Ile-. The H sequence, especially after the aspartyl residue at position 11, is rich in hydrophobic residues, consistent with the possibility that this section of the polypeptide chain is located within the membrane. The L sequence is hydrophilic near the amino terminus and then becomes moderately hydrophobic. The M sequence is of average polarity.     

Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.     

The complement binding-like domains of the murine homing receptor facilitate lectin activity

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality.