Expression and role of retinoic acid receptor alpha in lens regeneration

The role of retinoids in eye development has been well studied. Retinoids and their receptors regulate gene expression and morphogenesis of the eye. In this study, a highly specific antagonist of retinoic acid receptor (RAR)-alpha was used in an attempt to study its function in lens regeneration. It was found that this antagonist inhibited lens regeneration and lens fiber differentiation. It was also shown that RAR-alpha is expressed in the lens during the process of regeneration. These results indicate that different RAR might have unique as well as redundant effects and patterns of expression in the regenerating lens.     

Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.0 and 8.7. The mutant enzyme H216N has a maximal k(cat) at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. (18)O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2-HCO3- exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.     

Sutural loosening and skeletal flexibility during growth: determination of drop-like shapes in sea urchins

The shape of sea urchins may be determined mechanically by patterns of force analogous to those that determine the shape of a water droplet. This mechanical analogy implies skeletal flexibility at the time of growth. Although comprised of many rigid calcite plates, sutural collagenous ligaments could confer such flexibility if the sutures between plates loosened and acted as joints at the time of growth. We present experimental evidence of such flexibility associated with weight gain and growth. Over 13-, 4-, and 2-week periods, fed urchins (Strongylocentrotus droebachiensis) gained weight and developed looser sutures than unfed urchins that maintained or lost weight. Further, skeletons of fed urchins force-relaxed more than did those of unfed urchins and urchins with loose sutures force-relaxed more than those with tight sutures. Urchins (Strongylocentrotus franciscanus) fed for two and a half weeks, gained weight, also had looser skeletons and deposited calcite at sutural margins, whereas unfed ones did not. In field populations of S. droebachiensis the percentage having loose sutures varied with urchin diameter and reflected their size-specific growth rate. The association between feeding, weight gain, calcite deposition, force relaxation and sutural looseness supports the hypothesis that urchins deform flexibly while growing, thus determining their drop-like shapes.     

Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California

The sensitivity and specificity of the microscopic agglutination test (MAT) as a method for detection of exposure to Leptospira spp. in California sea lions (Zalophus californianus) were determined. Sera came from individuals that demonstrated clinical signs of renal disease, had lesions suggestive of leptospirosis at necropsy, and had visible leptospires in silver stained kidney sections as positive controls. Sera from unexposed captive individuals were used as negative controls. The test was 100% sensitive at 1:3,200 for confirming renal infection and 100% specific at negative < 1:100 for detection of Leptospira interrogans scrovar pomona antibodies by MAT in California sea lions. Leptospira interrogans serovar pomona was used as a screening serovar because it has been isolated previously from the kidneys and placentas of California sea lions, and there appears to be cross-reactivity between serovar pomona and other serovars. Sera from 225 free-ranging California sea lions presented to one of three participating California (USA) coastal marine mammal rehabilitation centers in 1996 were then evaluated for antibodies to serovar pomona using the MAT. The overall seroprevalence was 38.2% (86/225), although the prevalence varied among locations from 100% (38/38) in animals at the Marine Mammal Care Center (Fort MacArthur, California, USA) to 0% (0/14) at SeaWorld California (San Diego, California). At The Marine Mammal Center (Sausalito, California) [prevalence 27.8% (48/173)], the majority of seropositive animals were subadults and adults, and males were 4.7 times more likely to be seropositive to serovar pomona than females. When combining results from all three centers, subadult and adult animals were more likely to be seropositive than pups and juvenile sea lions, and the highest proportion of seropositive animals presented during the autumn months. Serum elevations of blood urea nitrogen, creatinine, phosphorus, and/or calcium were associated with seropositivity to serovar pomona. We found no association between potassium or sodium levels and seropositivity.     

Nuclear RNA turnover

In mammalian cells, significantly more RNA is turned over in the nucleus than in the cytoplasm. However, only recently have we begun to understand the mechanisms and regulation of nuclear RNA decay.     

Microbial biohydrogenation of oleic acid to trans isomers in vitro

Ruminant products are significant sources of dietary trans fatty acids. Trans fatty acids, including various conjugated linoleic acid isomers, have been shown to act as metabolic modifiers of lipid metabolism. Trans fatty acids originate from biohydrogenation of dietary unsaturated fatty acids by gut microbes; however, the exact synthetic pathways are unclear. It was our goal to examine the biohydrogenation pathway for oleic acid, where oleic acid is hydrogenated directly to stearic acid. Our objective in this study was to trace the time course of appearance of 13C in labeled oleic acid to determine if trans monoenes are formed from the 13C-labeled oleic acid or if the 13C appears only in stearic acid as described in reviews of earlier work. Enrichments were calculated from the mass abundance of 13C in major fatty acid fragments and expressed as a percentage of total carbon isotopomers. Significant 13C enrichment was found in stearic acid, oleic acid, trans-6, trans-7, and in all trans C18:1 in positions 9-16. We concluded that the biohydrogenation of oleic acid by mixed ruminal microbes involves the formation of several positional isomers of trans monoenes rather than only direct biohydrogenation to form stearic acid as previously described.     

Pathophysiological contributions of fucosyltransferases in renal ischemia reperfusion injury

Ischemia reperfusion injury (IRI) is a major cause of delayed graft function. Recent studies have shown that selectins play an important role in IRI. Selectins bind to sialylated and fucosylated sLe(x) receptors, and two enzymes, fucosyltransferase IV (FucT-IV) and VII (FucT-VII), are important in the function of these receptors. We hypothesized that fucosyltransferase (FucT) enzymes were important pathophysiologic mediators of renal IRI. We therefore evaluated renal IRI in mice deficient in FucT-IV, FucT-VII, and both FucT-IV and FucT-VII and compared their renal function, tubular injury, selectin ligand expression, and neutrophil infiltration to those in wild-type control mice. Bilateral 30-min renal IRI was performed, and the results demonstrated that mice deficient in both FucT-IV/FucT-VII were significantly protected from renal IRI at 24 and 48 h compared with wild-type control mice. FucT-IV-deficient mice showed only modest protection from renal injury at 24 h. However, FucT-VII-deficient mice had similar injury as wild-type mice. Histological analysis of kidney tissue postischemia revealed that mice deficient in both FucT-IV and FucT-VII had significantly reduced tubular injury compared with wild-type mice. Selectin ligand expression increased postischemia in wild-type, but not FucT-IV/FucT-VII-deficient, mice. Neutrophil infiltration in postischemic kidneys of FucT-IV/FucT-VII-deficient mice was also attenuated. These data demonstrate that fucosyltransferases are important in the pathogenesis of renal IRI and are potential therapeutic targets.     

MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.