Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli

The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1. The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB. It appeared that rapid haem-haem electron transfer could still occur. The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.     

Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B

BACKGROUND: About 65 percent of previously untreated adults with primary acute myeloid leukemia (AML) enter complete remission when treated with cytarabine and an anthracycline. However, such responses are rarely durable when conventional postremission therapy is administered. Uncontrolled trials have suggested that intensive postremission therapy may prolong these complete remissions. METHODS: We treated 1088 adults with newly diagnosed AML with three days of daunorubicin and seven days of cytarabine and randomly assigned patients who had a complete remission to receive four courses of cytarabine at one of three doses: 100 mg per square meter of body-surface area per day for five days by continuous infusion, 400 mg per square meter per day for five days by continuous infusion, or 3 g per square meter in a 3-hour infusion every 12 hours (twice daily) on days 1, 3, and 5. All patients then received four courses of monthly maintenance treatment. RESULTS: Of the 693 patients who had a complete remission, 596 were randomly assigned to receive postremission cytarabine. After a median follow-up of 52 months, the disease-free survival rates in the three treatment groups were significantly different (P = 0.003). Relative to the 100-mg group, the hazard ratios were 0.67 for the 3-g group (95 percent confidence interval, 0.53 to 0.86) and 0.75 for the 400-mg group (95 percent confidence interval, 0.60 to 0.94). The probability of remaining in continuous complete remission after four years for patients 60 years of age or younger was 24 percent in the 100-mg group, 29 percent in the 400-mg group, and 44 percent in the 3-g group (P = 0.002). In contrast, for patients older than 60, the probability of remaining disease-free after four years was 16 percent or less in each of the three postremission cytarabine groups. CONCLUSIONS: These data support the concept of a dose-response effect for cytarabine in patients with AML who are 60 years of age or younger. The results with the high-dose schedule in this age group are comparable to those reported in similar patients who have undergone allogeneic bone marrow transplantation during a first remission.     

Isolation of Shigella dysenteriae serotypes 11, 12, and 13 from patients with diarrhea in Bangladesh

Nine isolates of bacteria biochemically resembling Shigella dysenteriae but not belonging to the 10 recognized serotypes were isolated from patients with diarrhea in Bangladesh. Further studies suggested that two, one, and six isolates belonged to the recently recognized S. dysenteriae serotypes 11, 12, and 13, respectively.     

Correlates of osteopenia in patients with cystic fibrosis

The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes.     

Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome involving a predisposition to cancers of the colon, endometrium and several other extra-colonic sites, accounting for approximately 1-5% of all colorectal cancer cases. It is not easily recognized because of a lack of distinctive clinical markers, making diagnosis and management of this disease problematic. To provide a basis for uniformity in diagnosis of HNPCC, the Amsterdam criteria were proposed and are currently in use. More recently, the discovery of four human mismatch repair genes (hMSH2, hMLH1, hPMS1 and hPMS2) has provided novel insight into the genetic basis of this disease, and raised the possibility of genetic diagnosis for management of HNPCC patients and their family members. This report summarizes the clinicopathologic aspects of HNPCC, reviews the recent genetic findings and surveillance strategies, and suggests a novel designation of certain patients as suspected HNPCC.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Clinical and health care cost consequences of infective endocarditis in mitral valve prolapse

In 1990-1991, a national survey was conducted to estimate the prevalence of Salmonella species among Canadian commercial turkey flocks. Two hundred and seventy flocks were randomly selected across Canada. The proportion sampled from each province was selected according to each province's share of the national turkey market. Samples, consisting of 12 pooled litter and four pooled dust samples, were used to determine the Salmonella status of the environment of each flock. Additionally, a one kilogram sample of feed was taken from each flock premise. Salmonella was recovered from environmental samples in 234/270 (86.7%) of flocks and from feed samples in 26/266 (9.8%) of flocks. Forty-eight different Salmonella serovars were isolated from flock environmental samples. The most prevalent serovars were S. anatum, S. hadar, S. agona, S. heidelberg and S. saintpaul which were isolated from 53/270 (19.6%), 49/270 (18.1%), 49/270 (18.1%), 42/270 (15.6%) and 34/270 (12.6%) flocks, respectively.