Enzymes of cyclic nucleotide degradation in eye tissues during experimental ocular herpes

Activity of phosphodiesterases disintegrating cAMP and cGMP in the cornea, sclera and ciliary body was investigated in health and in different stages of experimental herpetic keratitis. The problems concerning the role of the cyclase system in the pathogenesis of herpetic keratitis and the possibility of applying some of the drugs to the disease treatment are discussed.     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

Characterization of non-transferrin-bound iron clearance by rat liver

Recent evidence suggests that the hepatic iron-loading characteristic of hemochromatosis may result in part from efficient hepatic clearance of non-transferrin-bound iron, which is increased in this disorder. However, this hypothesis assumes that hepatic clearance remains highly efficient despite excess iron stores. We therefore studied hepatic uptake of non-transferrin-bound iron in the single-pass perfused rat liver under varying conditions. Animals were iron loaded or depleted by dietary manipulation, but no changes in the efficiency of ferrous iron uptake or the kinetic parameters were seen (single-pass extraction, 59-74%; Km, 16-19 microM; Vmax, 30-32 nmol X min-1 X g liver-1). Added divalent zinc, cobalt, and manganese ions reversibly inhibited ferrous iron uptake and the inhibition by zinc was shown to be competitive. Uptake required calcium, was markedly temperature-sensitive (delta E = 14.3 Kcal/mol), and was relatively insensitive to inhibition of cellular energy metabolism. Particles consistent with ferritin cores were seen in lysosomes of hepatic parenchymal cells within 30 min of perfusion with ferrous iron. These results suggest that ferrous iron is cleared from plasma by a passive, saturable transport process that is not regulated by the iron content of the liver and that may be shared with other transition metal ions. Because clearance is highly efficient, increased levels of non-transferrin-bound iron in plasma may present the liver with an obligatory iron load resulting in progressive accumulation and toxicity.     

用定点突变技术研究转录终止——rpoBC操纵子上~tL7茎环结构的作用研究

 E.coli RNA聚合酶ββ'亚单位编码的基因rpoBC与核糖核蛋白基因rplJL共同构成rpoBC操纵子。rpl-rpo间区转录终止信号~tL7的转录产物RNA有两组富含C-G的反向对称结构及一串寡聚U;反向对称区可形成1:2和3:4茎环或单一5:6茎环。 用M13mp11噬菌体插入~tL7的112bp片段重组M13mp11-490,在此基础上用定点突变技术建立~tL7的七核苷酸缺失突变体,从而破坏1:2茎环,建成M13mp11-85。 分别把原型~tL7及缺失型~tL7~v克隆到表达质粒pHR24中,构建成pHR37(P_ftsQ~tL7-galk)及pHR24-9(p_ftsQ-~tL7~v-galk)。测定galk基因产物半乳糖激酶活性,推算转录的终止效率。结果表明:~tL7终止效率为50％,~tL7~v为20％。说明仅有3:4茎环及寡聚U,即具终止作用; 1:2茎环通过某种方式加强3:4茎环从而提高终止效率。     

Fine needle aspiration diagnosis of Penicillium marneffei infection

A disseminated infection with Penicillium marneffei, a rare human pathogen that may infect both healthy and immunocompromised patients, was diagnosed by fine needle aspiration cytology in a patient infected with the human immunodeficiency virus. The presence of yeast-form organisms with an eccentric or central dot and occasional septate and elongated forms highly suggested the diagnosis, which was confirmed on culture. Establishment of the diagnosis is important because this infection is potentially curable.     

Neutrophils-induced increase of adenosine triphosphate depletion in rat neonatal cardiac myocytes with impaired energy metabolism.

Isolated, cultured rat neonatal cardiac myocytes were placed in medium supplemented with mitochondrial respiratory inhibitor potassium cyanide which caused a rapid adenosine triphosphate (ATP) depletion. These myocytes with the impaired energy metabolism ("hypoxia-like state") were exposed to unstimulated human neutrophils. Effect of human neutrophils on the myocytes in the "hypoxia-like state" was quantified as a total change in the amount of ATP in cardiac cells. After 5 hours of incubation of neutrophils with the myocytes in the "hypoxia-like state" an additional decrease (of 50 per cent) in ATP content was observed. Since catalase (which destroys hydrogen peroxide) prevented the further decline in ATP level in the myocytes with impaired energy metabolism, it seem that hydrogen peroxide and possibly their products are responsible for this effect. These results suggest that unstimulated human neutrophils after activation by the contact with injured cardiac cells caused further decrease of ATP level in target cells.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.