Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.Abbreviations cDNA complementary DNA - TLC thin-layer chromatography We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Prevalence of risk factors for cardiovascular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992

BACKGROUND: By 2016, the proportion of Canadians older than 65 years of age will increase to 16%, and there will be an increase in the absolute number of cases of cardiovascular disease in older Canadians. The Canadian Heart Health Surveys database provides information about this population upon which health policy related to cardiovascular disease can be based. This paper presents for the first time population-based data on the risk factors for cardiovascular disease in older Canadians. METHODS: Canadians from all 10 provinces participated in surveys of cardiovascular risk factors; health insurance registries were used as sampling frames. In each province, probability samples of 2200 adults 18 to 74 years old not living in institutions, on reserves or in military camps were asked to participate in interviews and to undergo testing at clinics for major risk factors for cardiovascular disease. RESULTS: A total of 2739 men (response rate 70%) and 2617 women (response rate 66%) aged 55 to 74 years participated in the survey and also provided follow-up clinical measurements at the clinic. Overall, 52% of participants were hypertensive, 26% had isolated systolic hypertension, and 30% had a total blood cholesterol level of 6.2 mmol/L or greater. Rates of current smoking were lower in women than men (17% v. 22%). Overall, 87% of men and 78% of women who were current smokers smoked at least 10 cigarettes per day. Only slightly more than half of participants exercised at least once a week for at least 15 minutes, and almost half had a body mass index of 27 or greater. In only 4% was no major risk factor for cardiovascular disease detected. INTERPRETATION: Significant numbers of older Canadians have one or more major risk factors for cardiovascular disease. Many of these risk factors are amenable to modification.     

Circular dichroism and fluorescence spectroscopic analyses of a proline-rich glycoprotein from human parotid saliva

A proline-rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda-max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30% beta-form conformation was predicted. Circular dichroism of PRG failed to detect either poly-L-proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, beta-turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of the N-linked Asn residues participating in some type of beta-turn.     

Nuclear magnetic resonance spectroscopic and computer-stimulated structural analyses of a heptapeptide sequence found around the N-glycosylation site of a proline-rich glycoprotein from human parotid saliva.

The proline-rich glycoprotein from human parotid saliva has a common heptapeptide sequence around four of six N-glycosylation sites (Maeda, N., H. S. Kim, E. A. Azen, and O. J. Smithies, 1985, J. Biol. Chem., 20:11123-11130). A synthetic model of the heptamer protein sequence, NH2-Q(1)-G(2)-G(3)-N(4)-Q(5)-S(6)-Q(7)-CONH2, was examined by nuclear magnetic resonance (NMR) spectroscopy and the ECEPP/2-VAO4A (Empirical Conformation Energy Program for Peptides) energy minimization computer algorithm (Scheraga, H. A., 1982, Quantum Chemistry Program Exchange, 454; Powell, M. J. D., 1964, Quantum Chemistry Program Exchange, 60). The NMR spectrum was almost completely assigned in dimethylsulfoxide-d6 (DMSO), and the amide chemical shift temperature dependence, phi dihedral angles, and chi 1 rotamer populations elucidated. These data indicated that a significant population of the heptamer could exist as a type I beta-turn [4----1 between Q(5) and G(2)] and/or a type II' beta-turn [4----1 between (Q)5 and G(2) and/or a gamma-turn [3----1 between Q(5) and G(3)] with the amino acid chi 1 torsion angles weighted toward the gauche- conformation. Starting from these three possible conformations, the ECEPP/2-VAO4A rigid geometry energy minimization program was used to find the localized predominant in vacuo structures of this heptapeptide sequence. The type II' beta-turn conformation best fits the data based on internuclear hydrogen-bonding distances, minimum potential energy considerations, and the NMR parameters.     

Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions.

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.