Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo.     

Origin of the left anterior descending coronary artery from the pulmonary artery: An unusual cause of angina in a middle-aged woman

A 48-year-old woman was admitted to our institution with angina pectoris and a systolic murmur. At cardiac catheterization, she was found to have an anomalous origin of the left anterior descending coronary artery from the pulmonary trunk. There was also an associated atrial septal defect and a bicuspid aortic valve.     

Identification of noncollagenous basement membrane glycopolypeptides synthesized by mouse parietal entoderm and an entodermal cell line

Using two-dimensional gel electrophoresis, we have identified two noncollagenous basement membrane (BM) glycopolypeptides which are synthesized by the mouse teratocarcinoma-derived parietal yolk sac (PYS) cell line. These glycopolypeptides have molecular weights of about 200,000 and isoelectric points of about 5.6. Polypeptides with identical parameters are synthesized by the parietal entodermal cells of mouse embryos and are found in Reichert's membrane. Pluripotent embryonal carcinoma cells (ECC) synthesize considerable amounts of the two polypeptides, whereas the yield from nullipotent ECC is negligible. The treatment of nullipotent F9 cells with retinoic acid, which induces entodermal differentiation, activates the synthesis of these polypeptides. These results indicate that the two polypeptides can be used as markers of parietal entoderm differentiation.     

Characterization of glycerol nonutilizing and protoperithecial mutants ofNeurospora

Summary Mutants defective in polyol metabolism and/or in protoperithecial development were selected inNeurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed intoN. crassa to facilitate genetic analysis. One,glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, betweenad-1 andrib-1; the other,glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal toad-9. Another mutant,gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal toad-9.     

Functional and morphological characterization of isolated bovine adrenal medullary cells

Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.     

Analysis of Immunoprecipitated Surface Glycoproteins in Measles Virions and in Membranes of Infected Cells

Measles viral envelope proteins were immune precipitated from membranes of infected cells and from purified virus and analyzed by polyacrylamide gel electrophoresis. Under reducing conditions, specific precipitates contained two major polypeptide bands, designated virus glycopeptides 1 and 2 (VGP-1 and VGP-2). Both polypeptides appeared to be glycosylated, as indicated by their incorporation of [¹⁴C]glucosamine in infected cells. VGP-2 appeared as a single band in specific precipitates of infected cells and as a double band in precipitates of purified virus. Trypsin treatment of infected cells showed that reduced VGP-2 may be composed of two unrelated polypeptides. One may be F₁, which is unglycosylated, and the other may correspond to the proteolytic cleavage product of VGP-1, which is glycosylated. The relation of VGP-1 and VGP-2 to smaller surface antigens (X and Y) obtained by tryptic treatment of infected cells remains to be elucidated. In cells taken at various times postinfection and analyzed for viral membrane proteins, VGP-1 was detected at all times, indicating that the input virus VGP-1 was inserted into the cell and could not be differentiated from newly synthesized VGP-1. VGP-2 was not detectable before 24 h postinfection. In precipitates of cells 4 h postinfection and of infected cells incubated at pH 5.8, an additional polypeptide band migrated immediately ahead of VGP-1. We conclude that VGP-2 (molecular weight, 42,000) possibly consists of two components, one of which is the tryptic cleavage product of VGP-1 and the other of which is the unglycosylated polypeptide, F₁.     

Heavy Metal-Activated Synthesis of Peptides in Chlamydomonas reinhardtii

In this study, we have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd²⁺, Hg²⁺, and Ag⁺. Cells cultured in the presence of sublethal concentrations of Cd²⁺ synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 × 10³. The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg²⁺-treated cells, the principal thiol-containing compound induced by Hg²⁺ ions was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag⁺ ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd²⁺-induced peptides. But, in contrast to the results obtained using Cd²⁺ as an inducer, these molecules did not accumulate to significant levels in Ag⁺-treated cells. The presence of physiological concentrations of Cu²⁺ in the growth medium blocked the synthesis of the Ag⁺-inducible component(s) and rendered cells resistant to the toxic effects of Ag⁺, suggesting competition between Cu²⁺ and Ag⁺ ions, possibly at the level of metal uptake.     

Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)