Circular forms of Uukuniemi virion RNA: an electron microscopic study.

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3'' and 5'' ends of linear molecules.     

Drug induced changes in cardio-vascular parameters in the Atlantic cod,Gadus morhua

Summary Ventral (VAP) and dorsal (DAP) aortic blood pressure, heart rate (HR) and cardiac output ( ) were recorded simultaneously in unanaesthetized Atlantic cod, and the effects of vasoactive drugs on the cardio-vascular parameters studied. Mean resting values for the parameters were VAP=4,39 kPa, DAP=2,49 kPa, HR=41 beats/min, and = 29,1 ml/min×kg. Adrenaline constricted the systemic vasculature, dilated the branchial vasculature and caused a decrease of HR and due to a cholinergic reflex. After atropine pre-treatment this reflex was abolished, and the effect of adrenaline on blood pressure enhanced. A small decrease in persisted after atropine, presumably reflecting the effect of an increased end-systolic afterload.Phenylephrine produced a weak increase in systemic vascular resistance, while isoprenaline lowered both systemic and branchial vascular resistance. The effect of isoprenaline is probably mediated by beta adrenoceptors in both vascular beds, since propranolol antagonizes the effect.Acetylcholine in low doses produces a drop in without affecting HR, while higher doses also stop the heart. There is no significant change in either branchial and systemic vascular resistance after acetylcholine.Abbreviations VAP mean ventral aortic blood pressure - DAP mean dorsal aortic blood pressure - TBPD trans-branchial blood pressure drop - HR heart rate - SV stroke volume - cardiac output (ventral aortic blood flow) - VR _g branchial vascular resistance - VR _s systemic vascular resistance     

Structural complexity of defective-interfering RNAs of Semliki Forest virus as revealed by analysis of complementary DNA.

The 18S defective interfering RNA of Semliki Forest virus has been reverse transcribed to cDNA, which was shown to be heterogeneous by restriction enzyme analysis. After transformation to E.coli, using pBR322 as a vector, two clones, pKTH301 and pKTH309 with inserts of 1.7 kb and 2 kb, were characterized, respectively. The restriction maps of the two clones were different but suggested that both contained repeating units. At the 3' terminus, pKTH301 had preserved 106 nucleotides and pKTH309 102 nucleotides from the 3' end of the viral 42S genome. The conserved 3' terminal sequence was joined to a different sequence in the two clones, and these sequences were not derived from the region coding for the viral structural proteins. The DI RNAs represented by the two clones are generated from the viral 42S RNA by several noncontinuous internal deletions, since the largest colinear regions with 42S RNA are 320 nucleotides in pKTH301, and 430 and 340 nucleotides in pKTH309. All these fragments had unique RNase T1 oligonucleotide fingerprints, suggesting that they were derived from different regions of 42S RNA.     

Sympathetic nervous control of blood flow in the gills of the Atlantic cod,Gadus morhua

Summary The effects of sympathetic nerve stimulation, adrenaline and isoprenaline on the inflow pressure and efferent arterial and venous flow rates were studied in a cod gill preparation perfused at constant flow rate.The dominant effect of adrenaline was a reduced inflow pressure, accompanied by an increase in arterial flow and a decrease in venous flow. Isoprenaline also decreased the inflow pressure, but the changes in both outflow rates were small or absent.Sympathetic nerve stimulation gave arterial and venous flow changes comparable to the adrenaline effects, but the inflow pressure increased during nerve stimulation. Propranolol has little effect on the nerve responses, but phentolamine abolished or reversed the increase in inflow pressure, and also decreased or abolished the changes in outflow rates.The possible sites of action of the sympathetic fibres, and the distribution of adrenoceptors in the effector tissue is discussed. It is concluded that the main effect of sympathetic nerve stimulation is -adrenoceptor mediated, involving constriction of the arterio-venous pathway. The-adrenoceptor mediated control of total branchial vascular resistance may largely depend on circulating catecholamines.     

Isolation and characterization of cells from rat adipose tissue developing into adipocytes.

To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.     

A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase

The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1' position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1' position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1)

Amelogenesis imperfecta is characterized by the defective formation of tooth enamel. Here we present evidence that the X-linked form of this disorder (AIH1) is caused by a structural alteration in one of the predominant proteins in enamel, amelogenin. Southern blot analysis revealed a deletion extending over 5 kb of the amelogenin gene in males with the hypomineralization form of the AIH1. Carrier females were heterozygous for the molecular defect. The deletion appears to include at least two exons of the amelogenin gene and the extent of the deletion was verified by PCR analysis. The mutation was shown to segregate with the disease among 15 analyzed individuals belonging to the same kindred. Our results link a defect in the amelogenin gene to the abnormal formation of enamel. We thus conclude that the amelogenin protein has a role in biomineralization of tooth enamel.     

The spring development of phytoplankton in Lake Erken: species composition,biomass, primary production and nutrient conditions — a review

Relative abundance and within-lake distributions of three fishes, northern redbelly dace (Phoxinus eos), finescale dace (Phoxinus neogaeus), and central mudminnow (Umbra limi), were examined using minnow traps in Tuesday Lake, a small bog lake in the Upper Peninsula, Michigan. For these species, catches in minnow traps placed at the perimeter of the lake were 21 to 52 times higher than catches in midlake traps. Variance: mean ratios of perimeter trap catches indicated that both dace species were highly aggregated while the distribution of mudminnows was less aggregated or random. Over an 11 day period during which all fish caught were removed from the lake, catch per unit effort (CPUE) of both dace species declined in response to fish removal. In contrast, CPUE for mudminnows was low initially, increased to an asymptote and then declined only in the last 5 days of the fish removal. The patterns of CPUE for mudminnows indicated that mudminnow trapability and/or activity was reduced in the presence of high densities of dace. The low abundance of dace in traps with many mudminnows suggested mudminnows avoided traps already containing dace. Throughout the removal period, CPUE provided an accurate index of dace abundance, whereas this was true for mudmnnows only after dace populations had been reduced drastically. Therefore, in any use of minnow traps to estimate populations, both spatial distributions and relative species abundance of small fishes must be taken into account.