Covalent crosslinking of myosin subfragment-1 and heavy meromyosin to actin at various molar ratios: Different correlations between ATPase activity and crosslinking extent

Summary This paper describes a systematic study of crosslinking of skeletal muscle myosin subfragment-1 (S1) and heavy meromyosin (HMM) to F-actin in the rigor state with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). We followed the time courses of S1 or HMM head crosslinking at various actin:S1 or actin:HMM head molar ratios and the resulting superactivation of ATPase activity. The ATPase activity of the covalent complexes was measured at 0.5 M KCl, where the covalent complexes retain superactivated ATPase activity but the activity of uncrosslinked myosin heads is not activated by actin. S1 crosslinking was slowest at the actin:S1 molar ratio of 11, but faster at larger molar ratios, where more than 80% of added S1 could be crosslinked to actin. In spite of the dependence of crosslinking rate on actinS1 ratio, there were two linear correlations between ATPase activity and the extent of S1 crosslinking to actin: one for S1 crosslinked to actin at actinS1 molar ratios more than 2.71 and the other for S1 crosslinked at a molar ratio of 11. Extrapolation of the former correlation line to 100% crosslinked S1 gave an ATPase activity of 39 s^–1 for actin-S1 covalent complex at 25 °C, whereas that of the other correlation line gave 21 s^–1. The latter smaller activity suggests that the interface between actin and S1 in their rigor complexes at a molar ratio of 11 is different from that at molar ratios of more than 2.71. The acto-HMM crosslinking rate depended on the ratio of actin to HMM head, like that of S1 crosslinking to actin. The ATPase activity of crosslinked actin-HMM was, unlike that of actin-S1 covalent complexes, bell-shaped as a function of the crosslinked heads, but chymotryptic conversion of HMM to S1 in the covalent complexes made the bell-shaped characteristics disappear and increased the activity close to that of actin-Sl covalent complexes. These results indicate that some physical constraint imposed on myosin heads suppresses the actin-activated ATPase activity of HMM crosslinked to actin.     

大鼠催乳素瘤形成过程中催乳素基因的表达

给ＳＤ大鼠皮下埋植内含１７β－雌二醇硅橡胶管３０、６０、１２０天后，发现大鼠垂体重量随给药时间延长呈持续性增加；用放射免疫法测定血浆催乳素含量，亦依次明显升高；用Ｎｏｒｔｈｅｒｎ印迹杂交方法检测垂体细胞中ＰＲＬ基因，发现ＰＲＬ ｍＲＮＡ含量也依次显著增加，但其增加却远低于血浆ＰＲＬ含量的增加，提示β－雌二醇除了促进ＰＲＬ基因的转录外，还可能增加ＰＲＬ ｍＲＮＡ的翻译效率。     

Changing seroepidemiology of hepatitis B, C, and D virus infections in high-risk populations

Needle-sharing and sexual contact are important transmission routes of hepatitis B, C, and D virus (HBV, HCV, HDV) infection. This study aimed to investigate the current status of these viral infections among high-risk populations including prostitutes and intravenous (i.v.) drug users, compared with the prevalence rate reported previously to examine the changing seroepidemiology. Of the 916 female prostitutes, 79 (9%) were positive for antibody to HCV (anti-HCV), 111 (12%) were positive for HBV surface antigen (HBsAg), and 5 (5%) had antibody to HDV (anti-HDV). The prevalence rate was significantly lower compared to that in 1989-1991 (12%, P = 0.037) for HCV infection, and to that in 1988 (59%) and 1996 (40%) (P < 0.0001) for HDV infection. Of the 494 i.v. drug users, 87 (18%) patients were HBsAg carriers and 12 (14%) were anti-HDV-positive. The prevalence rate of HDV infection was significantly lower than that reported in 1985 (79%, P < 0.0001). Among the 443 tested i.v. drug users, 182 (41%) were anti-HCV-positive, significantly lower than that in 1985 (53%, P = 0.026). Of the 263 male prostitutes, 11 (4%) were anti-HCV-positive, 45 (17%) were HBsAg-positive, and 7 (16%) were anti-HDV-positive. Of the 129 illegal immigrant prostitutes, 7 (5%) were anti-HCV-positive, 15 (12%) were HBsAg-positive and none were positive for anti-HDV. In conclusion, the findings indicate a declining prevalence of HCV and HDV infections among drug users and prostitutes over the past 16 years. Male prostitutes and immigrant prostitutes are new "high-risk" populations and may become a reservoir for disease transmission.     

beta2-adrenergic receptor gene polymorphisms in myasthenia gravis (MG)

The beta2-adrenergic receptor (beta2-AR) belongs to the group of G-protein coupled receptors and is present mainly on skeletal and cardiac muscle cells and lymphocytes. The gene encoding beta2-AR (ADRB2) displays a moderate degree of heterogeneity in the human population. The distribution of polymorphisms at amino acid positions 16, 27 and 164 is changed in asthma, hypertension and obesity. We have earlier reported a decreased density of the beta2-AR on peripheral blood mononuclear cells and the presence of beta2-AR antibodies in patients with MG. Since certain polymorphisms affect the function of the beta2-AR, it was of interest to analyse these in MG. Using allele-specific polymerase chain reaction amplification, we revealed an over-representation of homozygosity for Arg16 and a lower prevalence of homozygosity for Gly16 in MG patients compared with healthy individuals. The increased frequency of homozygosity for Arg16 was due to a contribution from patients with generalized MG but not from patients with only ocular disease. Homozygosity for Glu27 was negatively associated with both the presence of beta2-AR antibodies and severity of disease. Moreover, acetylcholine receptor (AChR) antibodies were more often present in patients being homozygous for Gln27. Our results imply that homozygosity for Arg16 confers susceptibility to generalized MG, and that certain polymorphisms at amino acid position 27 are associated with subgroups of patients.     

Glial cell line-derived neurotrophic factor receptor alpha1 availability regulates glial cell line-derived neurotrophic factor signaling: evidence from mice carrying one or two mutated alleles

Glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1, also known as GDNFR-alpha) is a glycolipid-anchored membrane protein of the GFRalpha family, which binds glial cell line-derived neurotrophic factor [Jing S. et al. (1996) Cell 85, 1113-1124; Treanor J. J. et al. (1996) Nature 382, 80-83], a survival factor for several populations of central and peripheral neurons, including midbrain dopamine neurons [Lin L. F. et al. (1993) Science 260, 1130-1132], and mediates its ligand-induced cell response via a tyrosine kinase receptor called Ret [Takahashi M. et al. (1988) Oncogene 3, 571-578; Takahashi M. and Cooper G. M. (1987) Molec. Cell Biol. 7, 1378-1385]. In this paper, we show that mice with a null mutation of the GFRalpha1 gene manifest epithelial-mesenchymal interaction deficits in kidney and severe disturbances of intestinal tract development similar to those seen with glial cell line-derived neurotrophic factor or Ret null mutations. There is a marked renal dysgenesis or agenesis and the intrinsic enteric nervous system fails completely to develop. We also show that newborn GFRalpha1-deficient mice display no or minimal changes in dorsal root and sympathetic ganglia. This is in contrast to the deficits reported in these neuronal populations in glial cell line-derived neurotrophic factor and Ret null mutations. Mesencephalic dopaminergic neurons in the substantia nigra and ventral tegmental area appear intact at the time of birth of the mutated mice. Mice homozygous for the GFRalpha1 null mutation die within 24 h of birth because of uremia. Heterozygous animals, however, live to adulthood. There is a significantly reduced neuroprotective effect of glial cell line-derived neurotrophic factor in such heterozygous animals, compared with wild-type littermates, after cerebral ischemia. Taken together with previous data on glial cell line-derived neurotrophic factor and Ret, our results strongly suggest that GFRalpha1 is the essential GFRalpha receptor for signaling in the glial cell line-derived neurotrophic factor-Ret pathway in the kidney and enteric nervous system development, and that GFRalpha2 or GFRalpha3 cannot substitute for the absence of GFRalpha1. Moreover, neuroprotective actions of exogenous glial cell line-derived neurotrophic factor also require full GFRalpha1 receptor expression.     

Effect of ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on mouse skin and in the newborn mouse

Ellagic acid, quercetin and robinetin were tested for theirability to antagonize the tumor-initiating activity of benzo[a]pyrene(B[a]P) and (±)-7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]P 7,8-diol-9,10-epoxide-2), the ultimate carcinogenic metaboliteof benzo[a]pyrene. Ellagic acid, robinetin or quercetin (2500nmol) had no tumor-initiating activity on mouse skin, but thetopical application of 2500 nmol of ellagic acid 5 min beforea tumor-initiating dose of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2caused a 59–66% inhibition in the number of skin tumorsper mouse that were observed after 15–20 weeks of promotionwith 12-O-tetradecanoylphorbol-13-acetate. Similar treatmentwith 2500 nmol of robinetin or quercetin caused a statisticallyinsignificant 16–24% inhibition in the tumor-initiatingactivity of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2 applied5 min later. Treatment of mice with 2500 nmol of ellagic acid5 min before the application of 50 nmol of B[a]P inhibited themean number of skin tumors per mouse by 28–33% after 15–20weeks of promotion, but these decreases were not statisticallysignificant. Robinetin and quercetin had little or no effecton the tumor-initiating activity of B[a]P on mouse skin. Treatmentof preweanling mice with 1/7, 2/7 and 4/7 of the total doseof ellagic acid (300 nmol), robinetin (1400 nmol), myricetin(1400 nmol) or quercetin (1400 nmol) i.p. on their first, eighthand fifteenth day of life, respectively, did not cause the formationof tumors in animals that were killed 9–11 months later.Similar treatment of preweanling mice with the above doses ofthe phenolic compounds 10 min before the i.p. injection of atotal dose of 30 nmol of B[a]P 7,8-diol-9,10-epoxide-2 duringthe animal's first 15 days of life caused a 44–75% inhibitionin the number of diol-epoxide-induced pulmonary tumors per mouse.Similar treatment with these plant phenols had little or noeffect on B[a]P-induced pulmonary tumors.     

Radiographic findings in cotton textile workers and the relationship to cigarette smoking

A group of 140 cotton textile workers from Shanghai, Shandong, Guangxi, and Beijing have had their chest radiographs taken using similar requirements. Most had come from the preparatory departments of cotton mills and had a history of exposure to cotton dust for at least 20 years. As controls, 140 healthy individuals with no dust exposure were matched with respect to sex, age, and smoking history. All the radiographs were read according to the ILO International Pneumoconiosis Classification, and the manifestations belonging to categories 0/0 and 0/1 were grouped as "normal," whereas categories 1/0, 1/1, and 1/2 and above as "abnormal." There was no significant difference in X-ray abnormalities between cotton textile workers and controls (P greater than 0.05). But when the data on the cotton textile workers and controls were combined, an appreciable difference in the incidence of radiographic abnormalities was found between smokers and non-smokers (P less than 0.001). Cotton dust exposure may induce nonspecific interstitial pulmonary changes, but these changes were exaggerated by cigarette smoking. These two factors appeared to have an additive effect on the pulmonary X-ray findings.     

Disposition of the naturally occurring antimutagenic plant phenol, ellagic acid, and its synthetic derivatives, 3-O-decylellagic acid and 3, 3'-di-O-methylellagic acid in mice

The effect of ellagic acid and some of its more lipophilk derivativeson the mutagenicity of (? )-7ß, 8-di-hydroxy-9 10-epoxy-7,8, 910-etrahydrobenz[a]pyrene was examined in Salmonella typhimuriumTA100. Ellagic acid, 3, 3' -di-O-methylellagic add, 4, 4' -di-O-methylellagicacid and 3-O-decyiellagic acid were found to have approximatelyequal antimutagenic activity. The tissue distribution and eliminationof ellagic add, 3, 3' -di-O-methyleCagic add and 3-O-decylellagicacid were examined in CD-I mice. Little or no ellagic acid (<1 nmol/g) was found in blood, lung or liver after the oral administrationby gavage of 300 µunol of ellagic acid per kg body weightor after feeding 1% of ellagic acid in the diet for 1 week.Following the i.p. administration of 120 µmol/kg of ellagicacid, the blood and lung levels of ellagic acid were 15–20nmol/g at 30 min after the dose, and the concentrations of ellagicacid decreased to 1–3 nmol/g at 6–8 h after thedose. A portion of the administered i.p. dose precipitated inthe abdominal cavity. After i.v. administration, ellagic acidwas eliminated very rapidly from blood, lung and liver, and 70% of the administered dose was recovered in the urine andfeces as free ellagic acid and its conjugates. At 2 h afteran i.v. injection of 60 µ/kg of ellagic add, 46% of thedose was recovered in the urine as ellagic acid and its conjugates.Of this amount, about half was excreted as free ellagic acidand half was excreted as conjugates. An additional 25% of thedose was recovered in the feces (mostly as free ellagic acid)after 7 h. The disposition of 3, 3' -di-O-methylelIagic acidor 3–O-decyIellagic acid after i.v. administration (32µmol;sol;kg) was examined and compared to the dispositionof the same i.v. dose of ellagic acid. The concentrations ofellagic acid, 3,3' -di-O-methylellagic acid and 3-O-decytellagicadd decreased rapidly in the blood, liver and lung, but theconcentrations of 3-O-decylellagic add in the lung throughoutthe experimental period (2–360 min) was on average 20-to 40-fold higher than the corresponding average concentrationsof ellagic acid or 3, 3' -di-O-methylellagic acid.     

842例工人、农民和战士最大呼气流量-容积曲线的测定

作者用“点值法”对成都市内和郊区842例从事轻、中、重劳动的健康工人、农民和战士作了MEFV曲线和常规肺功能测定,发现工人和农民在低肺容积时流量增加,在高、中肺容积时流量下降,并以农民最明显,重劳动工人次之,轻、中劳动工人较不明显,战士则相反。揭示不同的体力活动方式及生活条件可导致不同肺容积时的流量改变,以适应其代谢增强时的耗氧需求。