Mutational analysis of the Drosophila sister-chromatid cohesion protein ORD and its role in the maintenance of centromeric cohesion

The ord gene is required for proper segregation of all chromosomes in both male and female Drosophila meiosis. Here we describe the isolation of a null ord allele and examine the consequences of ablating ord function. Cytologically, meiotic sister-chromatid cohesion is severely disrupted in flies lacking ORD protein. Moreover, the frequency of missegregation in genetic tests is consistent with random segregation of chromosomes through both meiotic divisions, suggesting that sister cohesion may be completely abolished. However, only a slight decrease in viability is observed for ord null flies, indicating that ORD function is not essential for cohesion during somatic mitosis. In addition, we do not observe perturbation of germ-line mitotic divisions in flies lacking ORD activity. Our analysis of weaker ord alleles suggests that ORD is required for proper centromeric cohesion after arm cohesion is released at the metaphase I/anaphase I transition. Finally, although meiotic cohesion is abolished in the ord null fly, chromosome loss is not appreciable. Therefore, ORD activity appears to promote centromeric cohesion during meiosis II but is not essential for kinetochore function during anaphase.     

Myocardial infarction and regulatory myosin light chain

Myosin from cardiac muscle consists of two heavy chains and two pairs of light chain. Regulatory myosin light chain (RMLC) is phosphorylated by a Ca2+ and calmodulin dependent myosin light chain kinase. The impact of experimental myocardial infarction on cardiac RMLC was studied. The left anterior descending coronary artery of rabbits was ligated. Three, 7 and 14 days later the animals were euthanized, sections of the heart were frozen in liquid nitrogen and later subjected to 2-dimensional electrophoresis. Isoelectric focusing was carried out at a pH range of 4.5-5.4. Reproducible patterns of protein separation showed four spots with proteins of phosphorylatable regulatory light chains shifted to a more negative pH as compared to essential light chain. We investigated changes in phosphorylation of RMLC in infarcted heart muscle. As compared to sham operated animals, a decline in phosphorylation of RMLC was present in both infarcted and non-infarcted portions of the left ventricle; the latter was significant 7 days following the onset of ischemia. In contrast, the decline in percent phosphorylation in the infarcted area was not significant. The amount of RMLC decreased significantly in the infarcted portion. A highly significant reduction in the percent of viable cardiomyocytes accompanied the decline in phosphorylation. There was a significant correlation of RMLC following administration of isoproterenol, 7 and 14 days following onset of ischemia. Only faint traces of essential atrial myosin light chain (ALC-1) were present in the non-infarcted portion of the left ventricle. No correlation was found between percent phosphorylation and the amount of RMLC (density) following infusion of saline or isoproterenol. Isoproterenol significantly increased percent phosphorylation without altering the amount of RMLC protein. We conclude that myocardial infarction profoundly affects regulatory myosin light chain phosphorylation in the infarcted and non-infarcted areas of the myocardium and that RMLC plays a significant part in myocardial contractility.     

The N-terminal domain of human GABA receptor rho1 subunits contains signals for homooligomeric and heterooligomeric interaction

gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.     

Novel high-performance liquid chromatographic assay using fluorescence detection for the quantitation of plasma gamma-methylene-10-deazaaminopterin and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin, in patients with solid cancers in a phase I trial

Gamma-methylene-10-deazaaminopterin (MDAM), a unique dihydrofolate reductase inhibitor, has demonstrated antitumor activity against a broad spectrum of human solid tumors in preclinical studies. A novel reversed-phase, ion-pair high-performance liquid chromatography (HPLC) assay that uses fluorescence detection has been developed to quantitate levels of MDAM and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin (7-OH-MDAM), in human plasma. The recovery of MDAM and 7-OH-MDAM from plasma was >97% by a simple one-step deproteinization process using tetrabutylammonium bromide (TBABr) and methanol. MDAM and 7-OH-MDAM remained stable in plasma over a 28-day test period at ambient temperatures, and neither compound was light-sensitive. The limit of quantitation was 0.005 microM for both MDAM and 7-OH-MDAM. This assay has been found to be simple, sensitive and reproducible in determining plasma concentrations of MDAM and 7-OH-MDAM in patients with solid cancers in a phase I trial.     

Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues

A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.     

Heart rate variability in evaluation of the autonomic nervous system. A review

Analysis of heart rate variability is increasingly used for testing the function of the autonomic nervous system in cardiovascular disease and for the diagnosis of autonomic neuropathy. In cardiovascular disease, long-term data collection (several hours) is primarily used to describe the static activity in the different branches of the autonomic nervous system. In the diagnosis of autonomic neuropathy, short-term forced variations in heart rate are employed in order to describe the dynamic capacity of the parasympathetic nervous system. In the subsequent data-analysis several different principles may be used, and the purpose of this review is to describe these principles and their use in clinical and experimental settings.