Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%). This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.     

Analysis of Orthologous Retrovirus-Like Elements in the White-Footed Mouse,Peromyscus leucopus

Three loci in the genome of the white-footed mouse, Peromyscus leucopus, were examined for the presence or absence of orthologous copies of the retrovirus-like element mys using polymerase chain reaction. We examined these loci in 28 mice collected throughout the P. leucopus species range. Mys insertions were present in only one of the individuals examined at the mys-1 and mys-7 loci. Conversely, the mys-6 element was found in several individuals, but the presence of this element was limited to northern latitudes. Because the long terminal repeats (LTRs) of a given element are expected to be identical at the time of retrotransposition into the genome, and to accumulate changes over evolutionary time, within-element LTR sequence comparisons can be used to estimate the relative age of insertions. Within-element LTR differences are greater in mys-6 than in mys-1 or mys-7. The LTRs from orthologous mys-6 elements of six mice were sequenced. The alignment revealed 13 of the 22 differences between the right and left LTRs that were shared by all orthologous mys-6 sites, suggesting that relative to its time of insertion into the genome, mys-6 has only recently spread across the northern part of the species range. Received: 23 January 1996 / Accepted: 24 April 1996     

A test for preference of association in a color polymorphic poeciliid fish: laboratory study

Synopsis The ecological and evolutionary forces maintaining genetic polymorphism within populations is of continuing interest to evolutionary biologists. Male pygmy swordtails,Xiphophorus pygmaeus, are polymorphic at a Y-linked locus controlling body color. Fish with the + and cp alleles have blue bodies; those with the con allele have bright gold bodies. Male and female fish were tested in the laboratory to determine if there were any preferences for association with groups of male fish based on color. Single test fish were presented with two groups of males of different phenotypes, and times in which the test fish spent in proximity to each group were recorded. Males of different phenotypes showed no differences in preference, nor did phenotype of the males in the groups affect the behavior of the test fish. Females also showed no preference for males of a particular phenotype. Finally, female test fish showed no preference for groups containing a male with the rare phenotype compared to a group containing all males of the same phenotype.     

The ultrastructural basis of endothelial cell surface functions

Several enzymes, enzyme inhibitors, receptors and transport structures are situated on the luminal surface of endothelium. Some enzymes and transport systems are continuously active and, in effect, regulate the composition of blood moving downstream. Other components are latent. Their activities are not expressed in the absence of stimulus. Thus, endothelial cells injured by granulocytes or viral infection possess receptors for the Fc segment of IgG and for C3b, whereas normal endothelial cells do not. Both normal and injured endothelial cells express receptors for Clq. To visualize surface enzymes, inhibitors, receptors and transport structures, we have prepared surface replicas suitable for high resolution EM. The surface replication technique, coupled with immunocytochemical procedures, facilitates studies of the topographies of surface enzymes such as angiotensin converting enzyme (ACE) and carboxypeptidase N (CPN). In addition, the replicas provide unique views of the glycocalyx, a cell coating previously believed to be amorphous but now seen to be a highly organized carpet-work under which surface enzymes, receptors, etc are embedded. Given its content of fibronectin, the glycocalyx may be the Clq receptor. Cells treated with antibodies to ACE or CPN show disarrayed glycocalyces and bind Fc and C3b. Latency of the Fc and C3b receptors may be owing to the physical barrier provided by the glycocalyx. Apparently, damage to the glycocalyx creates conditions favoring binding of immune complexes, complement activation and intravascular coagulation with loss of gradients between blood and parenchyma. Whether some non-thrombogenic properties of endothelium require an intact glycocalyx deserves consideration as does the role of the glycocalyx in regulating microvascular permeability.     

Relationship between root volatiles of some carrot cultivars and their resistance to the carrot fly, Psila rosae

Field experiments investigated the resistance of some carrot cultivars to Psila rosae. In addition, headspace vapour and steam distillate from the roots of resistant and susceptible varieties were compared by gas-liquid chromatography. The field data confirmed that resistance may operate by decreasing the numbers of eggs laid indicating a nonpreference by the female Psila. Root resistance to the larva was also confirmed but the mechanism was unclear. A new finding was that root resistance is independent of the effect of egg laying, some cultivars evincing one or the other effect and some such as Regulus Imperial displaying both. It was clear that root resistance to the larva is the crucial prerequisite in breeding resistant varieties.One consistent difference was detected by the chemical comparisons: intact roots of resistant varieties released substantially less volatiles. Specifically, Regulus released almost five times less of the volatiles already shown to positively influence host-finding behaviour by the larva.

---

Résumé La résistance à P. rosae de quelques cultivars de carotte a été étudiée en plein champ. Parallèlement, les substances volatiles diffusées et celles extraites par la vapeur des racines de variétés résistantes et sensibles, ont été comparées en chromatographie gaz-liquide (GLC). Les résultats en champ ont confirmé que la résistance peut être due à une diminution du nombre d'oeufs pondus, révélant une absence d'attractivité pour les femelles de P. rosae. La résistance des racines aux larves a été aussi confirmée, mais les raisons n'en étaient pas claires. Un aspect nouveau est que la résistance des racines est indépendante de l'effet de la ponte, quelques cultivars présentant l'un ou l'autreeffet et certains, comme Regulus Imperial, manifestant les deux. Il est net que la résistance racinaire aux larves est la condition essentielle pour la sélection de variétés résistantes. Une différence importante a été mise en évidence par les comparaisons chimiques: les racines intactes de variétés résistantes libèrent nettement moins de substances volatiles. Précisément, Regulus a libéré 5 fois moins de substances volatiles déjà connues comme influençant positivement le comportement de découverte de l'hôte par la larve.

     

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans

Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.     

Studies in vitro of the nature and synthesis of the cell wall ofChlamydia trachomatis

The possibility thatChlamydia trachomatis contains peptidoglycan was examined by three methods. Preincubation of chlamydia with enzymes known to cleave peptidoglycan had no adverse effect on the subsequent development. Immunofluorescence studies with antistreptococcal peptidoglycan antisera failed to show any cross reactions with chlamydial antigens. The antichlamydial activity of anti-cell-wall antimicrobials was examined; lactams proved the most active, and cycloserine and bacitracin also showed antichlamydial activity. Alaphosphin, phosphomycin, and vancomycin showed no antichlamydial activity at the concentrations tested.     

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establisment of a monolayer from a low-density seed (7.5 × 10⁴ cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 × 10⁶ cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 × 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture I, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture I). Interdivision times (IDT) were longer and relatively constant in culture I until near confluence; none were < 10 h, whereas in 2, 24% of the IDT's were ≤ 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.