Effects of calmodulin and lipoxygenase inhibitors on LH (lutropin)- and LHRH (luliberin)-agonist-stimulated steroidogenesis in rat Leydig cells.

The results of this study, carried out with purified rat Leydig cells, indicate that there are no major differences in the stimulating effects of lutropin (LH) and luliberin (LHRH) agonists on steroidogenesis via mechanisms that are dependent on Ca2+. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. All three calmodulin inhibitors used (calmidazolium, trifluoperazine and chlorpromazine) were shown to block LH- and LHRH-agonist-stimulated steroidogenesis. This probably occurred at the step of cholesterol transport to the mitochondria. Similarly, three lipoxygenase inhibitors (nordihydroguaiaretic acid, BW755c and benoxaprofen), inhibited both LH- and LHRH-agonist-stimulated steroidogenesis. The amounts of the inhibitors required were similar for LH- and LHRH-agonist-stimulated steroidogenesis. Steroidogenesis stimulated by the Ca2+ ionophore A23187 was also inhibited, but higher concentrations of the inhibitors were required. Indomethacin (a cyclo-oxygenase inhibitor) increased LHRH-agonist-stimulated steroidogenesis;this is consistent with the role of the products of arachidonic acid metabolism via the alternative, lipoxygenase, pathway. The potentiation of LH-stimulated testosterone production by LHRH agonist was unaffected by indomethacin or by lipoxygenase inhibitors at concentrations that inhibited LH-stimulated testosterone production by 75-100%. It was not possible to eliminate a role of calmodulin in modulating the potentiation, although higher concentrations of the inhibitors were generally required to negate the potentiation than to inhibit LH- or LHRH-agonist-stimulated testosterone production.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]₂. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.     

Enterococcus faecalis infection in root canals – host‐derived or exogenous source?

Aims: Enterococcus faecalis is associated with a significant number of refractory endodontic infections. Previous studies report a prevalence of Ent. faecalis ranging from 24% up to 77% in teeth with failed endodontic treatment. The origin of the micro‐organism remains unclear, as enterococci do not belong to the normal oral microflora. The aim of this study was to determine whether these enterococci were of endogenous or exogenous origin. Methods and Results: Fifty consecutive patients with apical periodontitis in need of endodontic orthograde re‐treatment were included. Samples were collected from root canals, saliva and faeces and subjected to microbiological culturing. The genetic relationship between Ent. faecalis from root canals and isolates from the different host sources was determined using pulsed‐field gel electrophoresis. In 16% (8/50) of the patients, enterococci were collected from the root canal samples. The genetic analysis showed that the isolates from the root canals were not related to those from the normal gastrointestinal microflora. None of these patients had enterococci in their saliva samples. Conclusions: Endodontic infections with Ent. faecalis are probably not derived from the patient’s own normal microflora, which indicates that these infections ent. faecalis are of exogenous origin. Significance and Impact of the Study: This is the first study to genetically compare endodontic infectious Ent. faecalis isolates with isolates from the hosts’ own normal microflora.     

Qualitative shift to indirect development in the parasitic sea anemone Edwardsiella lineata

Direct development lies at 1 end of a continuum that encompassesvarious degrees of indirect development. Indirect developmentexists where a larval stage is interposed between the embryoand the adult and undergoes metamorphosis, though the ecologicaland morphological distinctiveness of the larval stage relativeto the adult stage can vary tremendously. There are numerousempirical examples where direct development has evolved fromindirect development, but little empirical evidence describinga recent transition from direct to indirect development. Here,we suggest 4 criteria for defining indirect, and therefore metamorphic,life histories. We then apply these criteria to address theplanula–polyp transition in cnidarians, focusing on 2species in the anthozoan family Edwardsiidae. The lined seaanemone, Edwardsiella lineata, has made a qualitative shifttowards indirect development that coincides with, and was potentiallyfacilitated by, the evolution of endoparasitism. We compareE. lineata's development with that of a closely related seaanemone, Nematostella vectensis, where the nonfeeding planulagradually develops the morphology of the adult polyp. In E.lineata, a novel parasitic life history stage is interposedbetween the planula and the polyp. We discuss how the evolutionof endoparasitism could facilitate the evolution metamorphiclife histories.     

Direct measurements of the kinematics and dynamics of bat flight

Experimental measurements and analysis of the flight of bats are presented, including kinematic analysis of high-speed stereo videography of straight and turning flight, and measurements of the wake velocity field behind the bat. The kinematic data reveal that, at relatively slow flight speeds, wing motion is quite complex, including a sharp retraction of the wing during the upstroke and a broad sweep of the partially extended wing during the downstroke. The data also indicate that the flight speed and elevation are not constant, but oscillate in synchrony with both the horizontal and vertical movements of the wing. PIV measurements in the transverse (Trefftz) plane of the wake indicate a complex 'wake vortex' structure dominated by a strong wing tip vortex shed from the wing tip during the downstroke and either the wing tip or a more proximal joint during the upstroke. Data synthesis of several discrete realizations suggests a 'cartoon' of the wake structure during the entire wing beat cycle. Considerable work remains to be done to confirm and amplify these results.     

Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.     

GENETIC AND ENVIRONMENTAL BASIS OF VARIABLE SEX RATIOS IN LABORATORY STRAINS OF POECILIOPSIS LUCIDA

Skewed sex ratios are common among several species of Poeciliopsis, a viviparous fish from northwestern Mexico. Since previous, unrelated studies from this laboratory (Angus and Schultz, 1983) suggested that deviation from a 1:1 sex ratio might be influenced by temperature, two inbred strains of P. lucida were tested for temperature-dependent sex determination by comparing sex ratios of offspring from pregnant females held at different water temperatures. Different sex ratios were produced by the two strains at the same temperature: one strain produced almost all-male offspring at 30°C and female-biased sex ratios at 24°C, while the other strain produced a 1:1 sex ratio at both temperatures. At intermediate temperatures, the labile strain produced sequentially fewer males with decreasing temperatures. The other strain produced a consistent sex ratio regardless of temperature. Poeciliopsis lucida apparently has a genetic polymorphism for temperature-influenced sex determination. An hypothesis is offered for the evolutionary origin of environmental sex determination.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.