Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.

Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2-deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh1-deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes. Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.

Integrating Clinical Data and Tear Proteomics to Assess Efficacy, Ocular Surface Status, and Biomarker Response After Orthokeratology Lens Wear.

Purpose: This study evaluated the efficacy and ocular surface status of Breath-O Correct, novel orthokeratology (OK) lenses, worn overnight for 3 months. Lens-induced changes in the tear proteome were evaluated. Methods: Thirty-one subjects, aged 19 to 26 years with refractive error from -1.00 to -5.00 D, were randomly assigned 1:1 to the treatment or control group. Refraction, visual acuity, corneal integrity, biomechanics and endothelial health, ocular surface changes, and subjective symptoms were assessed at the baseline, one-month, and three-month visits. The tear proteome was characterized over time using sequential window acquisition of all theoretical ion spectra mass spectrometry. Results: Lenses improved uncorrected visual acuity and reduced spherical powers with similar efficacy to other OK lenses. Significant reductions (P < 0.05) in corneal hysteresis (11.12 ± 1.12 to 10.38 ± 1.36 mm Hg) and corneal resistance factor (11.06 ± 1.32 to 9.90 ± 1.45 mm Hg) were observed in the treatment group after one month of lens wear, whereas other assessed factors remained unchanged. Thirteen and eight differentially expressed proteins were found after one month and three months of lens wear, respectively. Two proteins (proline-rich protein 27 and immunoglobulin V regions) were differentially expressed at both visits. Conclusions: Over a three-month period, Breath-O Correct lenses were overall safe, well tolerated, efficacious in refractive power reduction, and comparable with other OK lenses. Furthermore, their use caused only minor noninflammatory protein expression changes in the tear proteome. Translational Relevance: This study investigated the safety of orthokeratology contact lenses on the ocular surface in molecular aspects and standard clinical parameters.

Mantle plume helium in submarine basalts from the galapagos platform.

Helium-3/helium-4 ratios in submarine basalt glasses from the Galapagos Archipelago range up to 23 times the atmospheric ratio in the west and southwest. These results indicate the presence of a relatively undegassed mantle plume at the Galápagos hot spot and place Galápagos alongside Hawaii, Iceland, and Samoa as the only localities known to have such high helium-3/helium-4 ratios. Lower ratios across the rest of the Galápagos Archipelago reflect systematic variations in the degree of dilution of the plume by entrainment of depleted material from the asthenosphere. These spatial variations reveal the dynamics of the underlying mantle plume and its interaction with the nearby Galápagos Spreading Center.

Cloning and molecular characterization of the ontogeny of a rat ileal sodium-dependent bile acid transporter.

Sodium-dependent bile acid transport in the rat ileum is abruptly expressed at weaning. Degenerate oligonucleotides, based on amino acid sequence identities between the rat liver and hamster ileal transporters, were used to amplify a rat ileal probe. A 1.2-kb cDNA clone, which contains the full coding region (348 amino acids, 38 kD), was isolated by hybridization screening. In vitro translation yielded a 38-kD protein which glycosylated to 48 kD. Sodium-dependent uptake of taurocholate was observed in oocytes injected with cRNA. Northern blot analysis revealed a 5.0-kb mRNA in ileum, kidney, and cecum. A 48-kD protein was detected in ileal brush border membranes and localized to the apical border of villus ileal enterocytes. mRNA and protein expression, which were negligible before weaning, increased dramatically at weaning. Nuclear transcription rates for the transporter increased 15-fold between postnatal days 7 and 28. The apparent molecular weight of the transporter also increased between days 19 and 28. In summary, the developmental regulation of the rat ileal sodium-dependent bile acid cotransporter is characterized by transcriptionally regulated increases in mRNA and protein levels at the time of weaning with changes in apparent molecular weight of the protein after weaning.

Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene.

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.

Return to training and competition after deep venous calf thrombosis.

A 25-yr-old female triathlete presented with 4 d of increasing left calf pain that started 1 d after an elective termination of pregnancy during the eighth week of gestation. She had been training with running, bicycling, and swimming but did not recall any injury to the calf muscle. Deep venous thrombosis of the calf and popliteal veins was diagnosed, and she was treated with intravenous heparin and oral warfarin anticoagulation. Her major concern was return to training and competition. A protocol was developed based on experience with less active patients and animal studies to allow a progressive return to training activities over 5 wk, followed by running in the sixth week. After release from the hospital, she accelerated the return-to-training protocol and progressed to running within 3 wk. She developed the post-phlebitic syndrome that resolved within 2 yr. The diagnosis and treatment of deep venous thrombosis are reviewed, with specific attention to the needs of athletic patients.

Ventricular Tachycardia in a Marathoner.

In brief Young marathoners who run 60 miles per week may appear to be in great shape. But episodes of palpitations and light-headedness may signify a cardiovascular problem. Tests can reveal just how deceiving appearances can be.

Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.

Comparative analysis of the ontogeny of a sodium-dependent bile acid transporter in rat kidney and ileum.

An apical sodium-dependent bile acid transporter (ASBT) has recently been cloned and characterized in the rat ileum. Northern and Western blotting revealed both the ASBT mRNA and protein in rat kidney. The coding sequence of the kidney transcript was found to be identical to the previously cloned ileal ASBT. Indirect immunofluorescence studies localized the ASBT protein to the apical membrane of the renal proximal convoluted tubule. Kinetic analysis of sodium-dependent taurocholate uptake using membrane vesicles revealed a similar Michaelis-Menten constant value for taurocholate in the kidney and intestine. ASBT protein and function were present in the kidney but not the ileum from 7-day-old rats. On postnatal day 7, there was a sevenfold increase in ASBT steady-state mRNA levels in the kidney relative to the ileum, yet nuclear run-on assays revealed that the nascent transcription rates at this age were virtually the same. This suggests that the difference in the neonatal expression of the ASBT gene in the kidney and ileum may be in part due to differences in mRNA stability.

Upper-mantle dynamics revealed by helium isotope variations along the southeast Indian ridge.

Helium isotope variations in igneous rocks are important for relating isotopic heterogeneity to convective mixing in the Earth's mantle. High 3He/4He ratios at many ocean islands, along with lower and relatively uniform values in mid-ocean-ridge basalts (MORBs), are thought to result from a well mixed upper-mantle source for MORB and a distinct deeper-mantle source for ocean island basalts. At finer scales, 3He/4He variations along mid-ocean ridges have been related to underlying mantle heterogeneity, but relationships between the scales of geochemical segmentation and mantle convection remain enigmatic. Here we present helium isotope data for MORB glasses recovered along approximately 5,800 km of the southeast Indian ridge, and develop an approach to quantitatively relate spatial variations in geochemical and geophysical parameters at the Earth's surface. A point-to-point correlation analysis reveals structure in the helium isotope data at length scales of approximately 150 and approximately 400 km that appears to be related to secondary convection in the underlying mantle.

Hepatic basolateral sodium-dependent-bile acid transporter expression in two unusual cases of hypercholanemia and in extrahepatic biliary atresia.

The recent cloning of a human sodium-dependent bile acid transporter (NTCP) permits analysis of its expression in human liver disease and investigation of potential primary defects in its expression. NTCP from normal human liver (NHL) was first characterized in detail. Northern blotting of RNA from NHL revealed a 1.8-kb NTCP transcript. Western blotting of crude NHL plasma membranes using a carboxyterminal antipeptide antibody showed that NTCP is a 39-kd polypeptide that is N-glycosylated to a final molecular weight of 56 kd. Indirect immunofluorescent analysis of NHL sections indicated that the NTCP protein is expressed on the basolateral surface of hepatocytes. We hypothesized that the clinical phenotype of a defect in NTCP might be hypercholanemia in the relative absence of liver disease. Accordingly, the coding region of the NTCP gene of two children with this phenotype was sequenced after reverse transcription/polymerase chain reaction (RT/PCR) amplification. No primary defects in the deduced NTCP amino acid sequence were found. Despite the extremely high serum bile salt levels (235 and 126 micromol/L) in these two patients, NTCP messenger RNA (mRNA) and protein expression were quantitatively normal, in contrast to the published observations in a rat model of cholestasis secondary to common bile duct ligation. Hepatic steady-state NTCP mRNA levels in a group of 23 pre- and postportoenterostomy biliary atresia patients were inversely related to total bilirubin, indicating that extrahepatic bile duct obstruction leads to down-regulation of NTCP mRNA levels, similar to that observed in rat common bile duct ligation. Therefore the lack of down-regulation in the two patients with hypercholanemia indicates that elevated serum bile salts are not sufficient to down-regulate NTCP expression, these two patients have abnormal responses to hypercholanemia, or these two patients have a defect in a gene other than NTCP that influences hepatic clearance of bile salts.