Two specific ribonucleoprotein fragments from rat liver 60S ribosomal subunits

K+-depleted 60S ribosomal subunits from rat liver were submitted to a mild treatment with ribonuclease T1. Ribonucleoprotein fragments could be separated on sucrose gradients only when the digested subunits were partially deproteinized with a high KCl concentration (0.6 M) which removed seven proteins more or less completely and 5S RNA. The RNA and protein content of each fragment has been characterized. The largest ribonucleoprotein enclosed two RNA fragments of about 950,000 and 750,000 daltons and all the salt-resistant proteins except L5. The smallest one enclosed protein L5 (with L11, L17 and L26 in small amounts) and a 67,000 RNA piece. The subsequent hydrolysis of the large ribonucleoprotein produced several other ribonucleoproteins. One of them has been fully characterized: it enclosed a 250,000 RNA fragment and protein L12 (with L11, L25 and L30 in smaller amounts).     

Effect of ribonuclease T1 on ribosomal subunits of rat liver.

The accessibility of 28S RNA within the ribosomal subunits to ribonuclease T1 was studied, in comparing results obtained after enzyme treatment of compact, K+ deficient 60S subunits and of EDTA-treated 60S subunits. RNA, extracted from the subunits, using a mixture of sodium dodecyl sulfate and phenol was analyzed on sucrose gradients. The RNA from active subunits was only degraded in high enzyme concentrations. In the K+ deficient subunits, RNA is more accessible since it breaks down into 6 well-defined fragments, sedimenting between 4S and 18.5S. Within the EDTA-subunits, there is no more protection of the RNA. In fact, it is degraded by weak enzyme concentrations, as is the free 28S RNA, giving heterogeneous fragments. Comparison of the melting curves of subunits and free 28S RNA showed that it is only in EDTA subunits that proteins do not stabilize the secondary structure of RNA. In the case of 40S subunits, the action of ribonuclease T1 combines with the action of the endogenous nuclease which makes the degradation process more difficult to analyze.     

Detection of ischemia-reperfusion cardiac injury by cardiac muscle chemiluminescence

Various methods have been used in the past to assess the implication of oxygen free radicals (OFR) in ischemia-reperfusion-induced cardiac injury. Luminol-enhanced tert-butyl-initiated chemiluminescence in cardiac tissue reflects oxidative stress and is a very sensitive method. It was used to elucidate the role of OFR in cardiac injury due to ischemia and reperfusion. Studies were conducted on perfused isolated rabbit hearts in three groups (n = 8 in each): I, control; II, submitted to global ischemia for 30 min; III, submitted to ischemia for 30 min followed by reperfusion for 60 min. The heart tissue was then assayed for chemiluminescence (CL); content of malondialdehyde (MDA), an indicator of OFR-induced cardiac injury; and activity of tissue levels of antioxidants [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)].The control values for left and right ventricular CL and malondialdehyde were 81.1 ± 15.4 (S.E.) and 182.4 ± 50.3 (S.E.), mv-min-mg protein^–1; and 0.024 ± 0.006 (S.E.) and 0.324 ± 0.005 (S.E.) nmoles-mg protein^–1 respectively. Ischemia produced an increase in the cardiac CL (3.3 to 4.4 fold) and MDA content (2 to 2.6 fold). Reperfusion following ischemia also produced similar changes in CL and MDA content. The control values for activity of left ventricular SOD, catalase, and GSH-Px were 45.77 ± 1.73 (S.E.) U-mg protein^–1 5.35 ± 0.51 (S.E.) K-10^–3-sec^–1-mg protein^–1, and 77.50 ± 7.70 (S.E.) nmoles NADPH-min^–1-mg protein^–1 respectively. Activities of SOD and catalase decreased during ischemia but were similar to control values in ischemic-reperfused hearts. The GSH-Px activity of left ventricle was unaffected by ischemia, and ischemia-reperfusion. GSH-Px activity of the right ventricle increased with ischemia, and ischemic-reperfusion.These results indicate that cardiac tissue chemiluminescence would be a useful and sensitive tool for the detection of oxygen free radical-induced cardiac injury.     

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between highrisk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patiens will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.     

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel

Summary A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.The authors would like to thank U. Heber for many helpful discussions. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 176, projects B3 and B7) and by the Fonds der Chemischen Industrie.     

Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging.

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.     

Core Particles of Hepatitis B Virus and Ground Squirrel Hepatitis Virus I. Relationship Between Hepatitis B Core Antigen- and Ground Squirrel Hepatitis Core Antigen-Associated Polypeptides by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Peptide Mapping

The relationships among the core antigen polypeptides of hepatitis B virus (HBV) and ground squirrel hepatitis virus (GSHV) were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. The major core antigen polypeptides of liver-derived HBV (p22) and GSHV (p20.5) shared 56% of the spots in their peptide maps. Comparison of hepatitis B core antigen (HBcAg) p19 or ground squirrel hepatitis core antigen (GSHcAg) p16.5 with their respective major polypeptides indicated that these components probably resulted from cleavage of the major polypeptide of each virus. Other polypeptides smaller than the major component of each virus were often faint on polyacrylamide gels and probably resulted from the cleavage or degradation of components larger than p22 of HBcAg or p20.5 of GSHcAg, since their peptide maps contained spots unique to these high-molecular-weight components. p26 of GSHcAg and p27.5 of HBcAg shared approximately two-thirds of the spots on their peptide maps with those of their respective major core polypeptides. Furthermore, p37.5 of GSHcAg and p40 of HBcAg shared about 60% homology with their respective major polypeptides, and also shared many of the spots that were unique to p26 of GSHcAg or p27.5 of HBcAg but were not found in the peptide map of their respective core antigen polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands larger than 40,000 daltons were variably present, and peptide mapping indicated that these were aggregates of various smaller core antigen-associated polypeptides. The results suggest that p40 of HBcAg and p37.5 of GSHcAg are the largest unique polypeptides in these core particles, and that they are encoded for by the genome of each virus. That a subset of the spots unique to p40 or p37.5 was also found in p27.5 of HBcAg or p26 of GSHcAg, respectively, as compared to the major core polypeptides, also suggests that p27.5 and p26 are unique proteins encoded by the genome of each virus. It is proposed that the core antigen gene of each virus is larger than that which would encode the major polypeptide of each virus, and that the genetic organizations of the core genes of HBV and GSHV are very similar.     

Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.

The recently described protein kinase activity in hepatitis B virus core antigen particles (Albin and Robinson, J. Virol. 34:297-302, 1980) has been demonstrated here in the liver-derived core particles of ground squirrel hepatitis virus. Both protein kinase activities were initially associated with DNA polymerase-positive heavy core particles in CsCl density equilibrium gradients and shifted to polymerase-negative cores during the course of purification. The major core-associated polypeptide of each virus was the dominant species labeled. A variable number of other polypeptide species were also labeled by this reaction. Tryptic peptide mapping of both major and minor phosphorylated polypeptides of each virus resulted in similar patterns, suggesting that many of the sites of phosphorylation were the same in the components of each core particle. Hydrolysis of these phosphorylated core particles revealed a major phosphoamino acid as serine and a minor phosphoamino acid as threonine. The products of the protein kinase reaction in both human hepatitis B and ground squirrel hepatitis virus core particles, then, share many characteristics. The possible function(s) of this protein kinase activity is discussed in the light of similarly characterized activities in other animal viruses.     

Copper,manganese, zinc,and cadmium in tissues from New Zealanders

Concentrations of Cu, Mn, Zn, and Cd were measured in 13 different tissues collected at autopsy from 55 New Zealanders, aged 1 week to 74 years. All analyses were done by atomic absorption spectrophotometry. In general, concentrations of Mn and Zn were similar to those reported elsewhere but Cu levels were slightly lower. Concentrations of Cd were low in all tissues except kidney. Median values were in accordance with those reported for other “unexposed” populations. A significant trend of increasing concentrations with age was found for Cu in cartilage, Zn in kidney cortex and medulla, and Cd in all tissues except bone, fat, and hair. Declines with age were observed for Cu in liver, aorta, and skeletal muscle, for Mn in heart, aorta, and cartilage and for Zn in lung and muscle. There were no obvious relationships between tissue trace element levels and cause of death assigned according to three groups: sudden accidental, cardiovascular, or respiratory.     

Neurohypophyseal peptide levels in CSF and plasma during passive avoidance behavior in rats

Levels of vasopressin (AVP), oxytocin (OXT), and neurophysin (NP) in CSF and plasma of rats were determined during acquisition and retention of passive avoidance behavior. None of the levels of neurohypophyseal peptides in CSF were changed either during the adaptation period, or during acquisition or the retention of this behavior. Moreover, no differences were found in hormone levels in CSF of the various groups of rats subjected to different shock intensities during the acquisition trial. The marked differences in individual latencies of nonavoiding rats, and the differences in latencies due to a different shock intensity applied during the learning trial were not reflected by changes in CSF hormone levels. Neither AVP nor NP levels in plasma were affected by the different shock intensities applied, when measured at 20 min after the learning trial. In contrast, a decrease in plasma OXT levels was observed after application of a shock intensity of 0.25 mA during the learning trial. During retention of the passive avoidance response plasma levels of AVP, OXT and NP were not different from the levels found in the nonshocked groups. It is suggested that under the conditions used in this study the CSF is apparently not involved in the distribution of neurohypophyseal peptides to their possible sites of behavioral action in the brain.