Functional characterization of the Tn5 transposase by limited proteolysis

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.     

Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation

This study investigates the significance of the glucose-6-phosphate dehydrogenase (G6PD) catalyzed oxidative and the transketolase (TK) catalyzed nonoxidative pentose cycle (PC) reactions in the tumor proliferation process by characterizing tumor growth patterns and synthesis of the RNA ribose moiety in the presence of respective inhibitors of G6PD and TK. Mass spectra analysis of 13C-labeled carbons revealed that these PC reactions contribute to over 85% of de novo ribose synthesis in RNA from [1,2-(13)C]glucose in cultured Mia pancreatic adenocarcinoma cells, with the fraction synthesized through the TK pathway predominating (85%). Five days of treatment with the TK inhibitor oxythiamine (OT) and the G6PD inhibitor dehydroepiandrosterone-sulfate (0.5 microM each) exerted a 39 and a 23% maximum inhibitory effect on cell proliferation in culture, which was increased to 60% when the two drugs were administered in combination. In vivo testing of 400 mg/kg OT or dehydroepiandrosterone-sulfate in C57BL/6 mice hosting Ehrlich's ascitic tumor cells revealed a 90.4 and a 46% decrease in the final tumor mass after 3 days of treatment. RNA ribose fractional synthesis through the TK reaction using metabolites directly from glycolysis declined by 9.1 and 23.9% after OT or the combined treatment, respectively. Nonoxidative PC reactions play a central regulating role in the carbon-recruiting process toward de novo nucleic acid ribose synthesis and cell proliferation in vitro and in vivo. Therefore, enzymes or substrates regulating the nonoxidative synthesis of ribose could also be the sites to preferentially target tumor cell proliferation by new anticancer drugs.     

Clinical and molecular heterogeneity in carbonic anhydrase II deficiency and prenatal diagnosis in an Italian family

Carbonic anhydrase (CA) II deficiency is characterized by osteopetrosis, renal tubular acidosis, cerebral calcification, and usually severe mental retardation. We describe an Italian boy with this disease whose mental retardation was relatively mild and whose renal tubular acidosis had only a distal component. A novel mutation of a gt-->tt change of splice donor site at the 5' end of intron 6 was demonstrated. Comparison of this patient with two previous Italian families with different mutations illustrates the clinical and molecular heterogeneity of this disease. The identification of the mutation in this family provided the opportunity for prenatal diagnosis in a subsequent pregnancy.     

Monocyte immune cell response and copper status in beef steers that grazed endophyte-infected tall fescue

A 3-yr study was conducted to evaluate immune response and Cu status of yearling beef steers as a consequence of grazing tall fescue (Festuca arundinacea Schreb.) infected (E+) with the endophyte fungus Neotyphodium coenophialum ([Morgan-Jones and Gams] Glenn, Bacon, and Hanlin). During a preliminary study in 1994, 24 weanling Angus and Angus x Hereford steers were blocked by breed and weight (initial BW 271 kg; SD 25) and were randomized to E+ and low endophyte (E-) fescue in pastures at Glade Spring, VA. Grazing began in April and was discontinued in July. In 1995 and 1996, 24 weanling Angus and Angus x Hereford steers (initial BW 249 kg, SD 20 and 240 kg, SD 15, respectively) were randomized to the E+ and E- pastures at Glade Spring during each year. Grazing began in April and continued until September in 1995 and October in 1996. In 1994, steers that grazed E+ fescue exhibited lower (P < .05) phagocytic activity, major histocompatibility complex (MHC) class II expression, ceruloplasmin, and serum Cu than steers that grazed E- tall fescue. During 1995, steers grazing E+ fescue had lower (P < .05) phagocytic activity and MHC class II expression than steers that grazed E- fescue. In 1996, one-half of the steers within each paddock received a Cu oxide bolus at the beginning of the grazing season. During 1996, phagocytic activity was lower (P < .01) and MHC class II expression tended (P < .07) to be lower in steers that grazed E+ tall fescue than in steers that grazed E- tall fescue. Copper supplementation increased (P < .05) MHC class II expression in July regardless of endophyte status over nonsupplemented steers. Steers that grazed E- tall fescue had higher (P < .05) plasma or serum Cu concentrations than steers that grazed E+ tall fescue in each year of the study. These data indicate that the endophyte compromised the immune function of grazing steers, and the data suggest a relationship with depressed Cu status.     

Postinfarction use of beta-blockers in elderly patients

beta-Adrenoceptor antagonists (beta-blockers) reduce mortality and recurrent myocardial infarction (MI) in older patients after both Q-wave MI and non-Q-wave MI. The effects of beta-blockers are to: (i) reduce complex ventricular arrhythmias, including ventricular tachycardia; (ii) increase the ventricular fibrillation threshold; (iii) reduce myocardial ischaemia; (iv) decrease sympathetic tone; (v) markedly attenuate the circadian variation of complex ventricular arrhythmias: (vi) abolish the circadian variation of myocardial ischaemia; and (vii) abolish the circadian variation of sudden cardiac death or MI. beta-Blockers reduce mortality in patients with MI and complex ventricular arrhythmias. In addition, they are excellent antianginal agents. Older persons with hypertension who have had an MI should be treated initially with a beta-blocker. beta-Blockers reduce mortality in patients with: (i) diabetes mellitus who have had an MI; (ii) MI and congestive heart failure with an abnormal or normal left ventricular ejection fraction; and (iii) MI and an asymptomatic abnormal left ventricular ejection fraction. Severe congestive heart failure, severe peripheral arterial disease with threatening gangrene, greater than first degree atrioventricular block, hypotension, bradycardia, lung disease with bronchospasm, and bronchial asthma are contraindications to treatment with beta-blockers.     

The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution

BACKGROUND: Base-excision DNA repair (BER) is the principal pathway responsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER pathway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzymes, glycosylase/lyases, also catalyze scission of the DNA backbone following base expulsion. Recent studies indicate that the glycosylase and lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink that facilitates DNA strand scission through imine (Schiff base)/conjugate elimination chemistry. The identity of this critical amine-bearing residue has not been rigorously established for any member of a superfamily of BER glycosylase/lyases. RESULTS: Here, we report the identification of the active-site amine of the human 8-oxoguanine DNA glycosylase (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active-site peptide irreversibly linked to substrate DNA to identify directly the active-site amine of hOgg1 as the epsilon-NH2 group of Lys249. In addition, we observed that the repair-inactive but recognition-competent Cys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by alkylation with 2-bromoethylamine, which functionally replaces the lysine residue by generating a gamma-thia-lysine. CONCLUSIONS: This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.     

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.     

Comparison of conventional anterior surgery and laparoscopic surgery for inguinal-hernia repair

BACKGROUND: Inguinal hernias can be repaired by laparoscopic techniques, which have had better results than open surgery in several small studies. METHODS: We performed a randomized, multicenter trial in which 487 patients with inguinal hernias were treated by extraperitoneal laparoscopic repair and 507 patients were treated by conventional anterior repair. We recorded information about postoperative recovery and complications and examined the patients for recurrences one and six weeks, six months, and one and two years after surgery. RESULTS: Six patients in the open-surgery group but none in the laparoscopic-surgery group had wound abscesses (P=0.03), and the patients in the laparoscopic-surgery group had a more rapid recovery (median time to the resumption of normal daily activity, 6 vs. 10 days; time to the return to work, 14 vs. 21 days; and time to the resumption of athletic activities, 24 vs. 36 days; P<0.001 for all comparisons). With a median follow-up of 607 days, 31 patients (6 percent) in the open-surgery group had recurrences, as compared with 17 patients (3 percent) in the laparoscopic-surgery group (P=0.05). All but three of the recurrences in the latter group were within one year after surgery and were caused by surgeon-related errors. In the open-surgery group, 15 patients had recurrences during the first year, and 16 during the second year. Follow-up was complete for 97 percent of the patients. CONCLUSIONS: Patients with inguinal hernias who undergo laparoscopic repair recover more rapidly and have fewer recurrences than those who undergo open surgical repair.