Isolation of sealed vesicles highly enriched with sarcolemma markers from canine ventricle

A highly enriched sarcolemma preparation was isolated by a combination of homogenizations and differential centrifugations of a homogenate of canine ventricular tissue followed by centrifugation of a membrane fraction layered over 24% (w/v) sucrose. The membrane fragments in the preparation were found to reside in a vesicular configuration by electron microscopy. Adenylate cyclase activity, specific ouabain binding sites, ouabain-sensitive potassium phosphatase activity and high-affinity (?)dihydroalprenolol binding sites were enriched from 27- to $\text{>40-}\text{fold}$ compared to the homogenate. 5′-Nucleotidase and antimycin A-insensitive NADH-cytochrome c reductase activities were enriched 10.1- and 3.0–3.9-fold, respectively, compared to the homogenate. Conversely, Ca²⁺-ATPase and succinic dehydrogenase activities were somewhat less than those expressed by the homogenate. The vesicles, or at least a fraction thereof, in the preparation were osmotically active as monitored by changes in 90° light scatter upon increases in the osmolarity of the extravesicular medium. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity in the preparation was 23.6 and 99.9 μmol/mg per h before and after 15 consecutive freeze-thaw cycles, respectively. This suggests that the preparation contains a large fraction of intact right-side-out vesicles but that about 24% of the vesicles in the preparation may be freely permeable. Conversely, the number of specific ouabain binding sites was increased about 1.4-fold by the freeze-thaw cycles which is consistent with the presence of     

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.     

High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus)

Serum samples of 400 white-tailed deer (Odocoileus virginianus) from 16 preserves in northeastern Illinois were tested for Neospora caninum antibodies in the N. caninum agglutination test using mouse-derived N. caninum tachyzoites and mercaptoethanol. Antibodies were found in 162 deer with titres of 1:40 (47 deer), 1:80 (32 deer), 1:160 (17 deer), 1:200 (eight deer), 1:400 (19 deer), 1:800 (17 deer) and > or = 1:1600 (22 deer). There were no significant differences in prevalence between age or sex of the deer. The high prevalence of N. caninum infection in deer is consistent with a sylvatic cycle of N. caninum.     

Vasomotor control in arterioles of the mouse cremaster muscle.

Recent advances in transgenic mouse technology provide novel models to study cardiovascular physiology and pathophysiology. In light of these developments, there is an increasing need for understanding cardiovascular function and blood flow control in normal mice. To this end we have used intravital microscopy to investigate vasomotor control in arterioles of the superfused cremaster muscle preparation of anesthetized C57Bl6 mice. Spontaneous resting tone increased with branch order and was enhanced by oxygen. Norepinephrine and acetylcholine (ACh) caused concentration-dependent vasoconstriction and vasodilation, respectively. Microiontophoresis of ACh evoked vasodilation that conducted along arterioles; the local (direct) response was inhibited by N(omega)-nitro-L-arginine (LNA), and both local and conducted responses were inhibited by 17-octadecynoic acid (17-ODYA). Microejection of KCl evoked a biphasic response: a transient conducted vasoconstriction (inhibited by nifedipine), followed by a conducted vasodilation that was insensitive to LNA, indomethacin, and 17-ODYA. Phenylephrine evoked focal vasoconstriction that did not conduct. Perivascular sympathetic nerve stimulation evoked constriction along arterioles that was inhibited by tetrodotoxin. These findings indicate that for arterioles in the mouse cremaster muscle, nitric oxide and endothelial-derived hyperpolarizing factor (as shown by LNA and 17-ODYA interventions, respectively) mediate vasodilatory responses to ACh but not to KCl, and that vasomotor responses spread along arterioles by multiple pathways of cell-to-cell communication.     

Effect of the esterase-D phenotype on its in vitro enzyme activity

Summary Esterase-D phenotypes and in vitro activity have been measured in red blood cells from 258 retinoblastoma patients and 73 unaffected relatives. Individuals with the 1-1 and 2-1 phenotypes showed distributions of enzyme activity which were not significantly different from each other. Individuals with the 2-2 phenotype, however, consistently showed a 25–30% lower level of enzyme activity. These results demonstrate the importance of determining the esterase-D phenotype in individuals with low ESD activity who might otherwise be assumed to carry a chromosome deletion at the esterase-D locus. We have also shown that, in vitro, the ESD enzyme is unstable over relatively short periods of time which, if uncontrolled, can give rise to a large variation in measured enzyme levels. The addition of b-mercaptoethanol to the assay buffer, which stabilises the enzyme, results in more consistent values being obtained within the same ESD phenotype. This feature could account in part for much of the variability in enzyme activity observed between different individuals in other studies.     

Studies on the interaction of Nile red with horseradish peroxidase in solution

The interaction of an extrinsic probe (Nile red) with an enzyme (horseradish peroxidase) in solution was investigated using fluorescence techniques. Nile red fluorescence is very environmentally sensitive and the presence of domains of differing polarity within the enzyme was ascertained by the decomposition of the Nile red emission spectrum. Further evidence for the position of the probe inside the enzyme was obtained from a molecular modeling study. A decrease in the emission intensity of the dye during incubation with horseradish peroxidase was explained by the occurrence of resonance energy transfer between the Nile red and the heme group in the enzyme. This was supported by a calculation of the critical transfer distance and a comparison of the fluorescence intensity of the dye in both the holo- and apo-enzyme. These data were then applied to the study of the effect of temperature on the structure of the enzyme, where changes in conformation were elucidated.     

The nature of mutation in Krabbe disease.

Galactosylceramide beta-galactosidase cross reacting material was demonstrated in brain, liver, and skin fibroblasts from patients with Krabbe disease. The mutant enzyme was antigenically identical to the normal enzyme and exhibited similar electrophoretic mobility. Normal quantities of the catalytically deficient enzyme were measured in the patients' tissues by a sensitive single radial immunodiffusion assay, indicating that the mutation is in structural gene for the enzyme protein.     

A rapid technique to predict malting quality in barley prior to harvest

Samples of grain from three spring barley cultivars of differing malting quality were collected at regular intervals during four weeks prior to harvest. The samples were dried, then assessed for relative grain hardness using the "Milling Energy" test. Ranking order of the cultivars for this character, which relates strongly to malting quality, was unaltered throughout. In a further experiment, it was demonstrated that selection for milling energy could be successfully practised on oven-dried grain collected six weeks after ear emergence.     

Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.     

Rhythms during extended dark periods determine rates of net photosynthesis and accumulation of starch and soluble sugars in subsequent light periods in leaves of Sorghum

Photosynthesis and photosynthate partitioning in leaves of Sorghum bicolor (L.) Moench exhibited a cyclic dependence on the duration (10–62 h) of dark periods inserted prior to bright light test periods (550 mol·s^-1·m^-2, photosynthetic photon flux). Maximum rates of net photosynthesis and of accumulation of starch and soluble sugars were, in the order given, two-, three- and fourfold greater than minimum values. Between 14 and 53% of photosynthate was retained in leaves depending on the length of the dark period. These changes were sufficient to account for the previously described stimulatory effect of short daylengths (i.e., long nights) on carbohydrate accumulation in leaves (N.J. Chatterton and J.E. Silvius, 1980, Physiol. Plant. 49, 141–144). The freerunning periods for the rhythmic dependence on darkness, determined either directly or by curve fitting, were about 24 h for net photosynthesis, 23 h for starch accumulation, and 26 h for solublesugar cccumulation. The deviation from period lengths of 24 h for carbohydrate accumulation indicates that these rhythms are probably endogenous and circadian. Initial maxima were observed after 14 h of darkness for photosynthesis, after 18–22 h for starch, and after 26 h for soluble sugars. The differences in period length and phase indicate that at least three separate rhythms underlie the dependence of photosynthate partitioning in Sorghum on darkness. Periods of low leaf dry-matter accumulation coincided approximately with periods of high net photosynthesis. As a result, maximum photoassimination and maximum export were synchronized and, furthermore, occurred at about the same time as expected light periods.Abbreviations and symbols DD (the nth h of) continuous darkness - LT Iocal time of day - free-running period length This paper is dedicated to Professor Wilhelm Nultsch on his 60 th birthdayMention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable