No evidence for ‘skewed’ inactivation of the X-chromosome as cause of Leber's hereditary optic neuropathy in female carriers

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder of the optic nerves. It has been proposed that the specific mutations in the mitochondrial DNA (mtDNA) that are associated with LHON require and X-chromosomally encoded permissive factor in order to become expressed. This would explain both the preponderance of male patients and the fact that most carriers of specific mtDNA mutations remain unaffected. Although linkage studies have been negative so far, the existence of such a factor has not been ruled out. We investigated the genealogical data of 24 large LHON pedigrees and concluded that the presumed X-linked factor would be recessively inherited and that at least 57% of the affected females would be heterozygous. Therefore, these females must be the victim of nonrandom X-chromosomal inactivation (skewed lyonization). However, analysis of X-chromosomal methylation patterns in 16 LHON-affected females revealed substantial skewing in only 15%–20% of cases, which is not significantly different from the patterns in 49 controls. Moreover, we found the frequency of LHON in daughters of affected heterozygous females to be twice to three times as high as in daughters of unaffected heterozygous females, which cannot be explained by an X-chromosomally inherited factor. We conclude that the results of our investigations do not support the hypothesis that LHON is a digenic disease with an X-linked factor being the main cause of loss of vision in the presence of relevant mtDNA mutations. Received: 1 June 1995 / Revised: 20 September 1995     

Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

3,6-Nonadien-1-ol from Citrullus vulgaris and Cucumis melo

cis,cis-3,6-Nonadien-1-ol isolated from watermelon and previously from muskmelon by vacuum steam distillation-extraction, was identified by int     

Hepatitis C virus genotypes in patients with chronic hepatitis C infection in southern Iran from 2016 to 2019

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The treatment of HCV infection has become more complicated due to various genotypes and subtypes of HCV. The treatment of HCV has made significant advances with direct-acting antivirals. However, for the choice of medicine or the combination of drugs for hepatitis C, it is imperative to detect and discriminate the crucial HCV genotypes. The main objective of this study was to determine the pattern of circulating HCV genotypes in southern Iran, from 2016 until 2019. The other aim of the study was to determine possible associations of patients’ risk factors with HCV genotypes. A total of 803 serum samples were collected in 4 years (2016–2019) from patients with HCV antibody positive results. A total of 728 serum samples were HCV-RNA positive. The prevalence of HCV genotypes was detected using the genotype-specific RT-PCR test for serum samples obtained from 615 patients. The HCV genotype 1 (G1) was the most prevalent (48.8%) genotype in the area, with G1a, G1b, and mixed G1a/b representing 38.4%, 10.1%, and 0.3%, respectively. Genotype 3a was the next most prevalent (47.2%). Mixed genotypes 1a/3a were detected in 22 (3.6%) and finally G4 was found in 3 (0.5%) patients. The other HCV genotypes were not detected in any patient. Genotype 1 (1a and 1b alone, 1a/1b and 1a/3a coinfections) is the most prevalent HCV genotype in southern Iran. HCV G1 shows a significantly higher rate in people under 40 years old.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.