Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase

Delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) precedes 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in steroid hormone metabolism. Both enzymes are members of the aldo-keto reductase (AKR) superfamily and possess catalytic tetrads differing by a single amino acid. In 3 alpha-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but a glutamic acid replaces His117 in 5 beta-reductase. By introducing the H117E point mutation into 3 alpha-HSD, we engineered 5 beta-reductase activity into the dehydrogenase. Homogeneous H117E 3 alpha-HSD reduced the double bond in testosterone to form 5 beta-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 microM and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 microM. These kinetic parameters were similar to those reported for homogeneous rat liver 5 beta-reductase [Okuda, A., and Okuda, R. (1984) J. Biol. Chem. 259, 7519-7524]. The H117E mutant also reduced 5beta-dihydrosteroids to 5 beta, 3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in kcat/Km versus wild-type 3 alpha-HSD. The ratio of 5 beta-reductase:3 alpha-HSD activity in the H117E mutant was approximately 1:1. Although the H117A mutant reduced Delta 4-3-ketosteroids, the 3 alpha-HSD activity predominated because the 5 beta-dihydrosteroids were rapidly converted to the 5 beta,3 alpha-tetrahydrosteroids. The pH-rate profiles for carbon-carbon double-bond and ketone reduction catalyzed by the H117E mutant were superimposable, suggesting a common titratable group (pKb = 6.3) for both reactions. In wild-type 3 alpha-HSD, the titratable group responsible for 3-ketosteroid reduction has a pKb = 6.9 and is assignable to Tyr55. The pH-rate profiles for 3-ketosteroid reduction by the H117A mutant were pH-independent. Our data indicate that Tyr55 functions as a general acid for both 3 alpha-HSD and 5 beta-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Delta 4-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5 beta-reductase by reorienting the substrate relative to the nicotinamide cofactor. This study provides functional evidence that utilization of modified catalytic residues on an identical protein scaffold is important for evolution of enzymatic activities within the same metabolic pathway.     

Simultaneous osseous genioplasty and meloplasty

We measured whether males of five species of poeciliid fish made detours to the right or left of a vertical-bar obstacle in order to approach a group of females. Three of these species, Gambusia holbrookiGambusia nicaraguensis and Poecilia reticulata showed a significant bias to the left, whereas Brachyrhaphis roseni and Girardinus falcatus showed a significant bias to the right. When tested for direction of turning in front of an opaque barrier, or when a dummy predator was used as a target in a detour test, G. holbrooki and G. falcatus showed similar biases to the right (opaque barrier) and left (predator), thus suggesting that the difference observed when females were used as a target could arise from species differences in the degree of sexual motivation in a novel environment. The two species that showed bias to the right with the females were less likely to exhibit sexual behaviour when placed in a novel environment. Moreover, manipulation of the factors affecting the relative strength of sexual motivation and of fear of a novel environment, such as how long fish were maintained in captivity or in the test apparatus before being tested, caused shifts in the direction of the lateral asymmetries. These results suggest that the presence of functional asymmetries in behaviour could be widespread among vertebrates and that the direction of such asymmetries tends to be strikingly similar in closely related species, thus supporting the hypothesis of an early evolution of laterality in brain and behaviour.Copyright 1997 The Association for the Study of Animal Behaviour1997The Association for the Study of Animal Behaviour     

rhDNase as an example of recurrent event analysis

We consider counting process methods for analysing time-to-event data with multiple or recurrent outcomes, using the models developed by Anderson and Gill, Wei, Lin and Weissfeld and Prentice, Williams and Peterson. We compare the methods, and show how to implement them using popular statistical software programs. By analysing three data sets, we illustrate the strengths and pitfalls of each method. The first example is simulated and involves the effect of a hidden covariate. The second is based on a trial of gamma interferon, and behaves remarkably like the first. The third and most interesting example involves both multiple events and discontinuous intervals at risk, and the three approaches give dissimilar answers. We recommend the AG and marginal models for the analysis of this type of data.     

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.     

Risk factors of infected pancreatic necrosis, its microbiology and antibiotic treatment

Acute pancreatitis is associated with greater and smaller necrosis in 20% of the cases. The lethality rate of sterile and infected necrosis is 10 and 15-40%, respectively. The results of a retrospective and a prospective study in acute pancreatitis have been analyzed in this study. Twenty patients suffering from infected necrosis due to acute necrotising pancreatitis were selected into the retrospective study. They were divided into two groups: Group 1 (N = 10) survivors, Group 2 (N = 10) those who died. The fate of patients was determined by their age, the severity of pancreatitis, and the effectiveness of the operation. In a prospective study 63 patients were operated due to benign pancreatic disease with fluid collection. Microbiological samples were taken during surgery in every case. It could be stated that the Enterobacteriaceae spp. play the principal role in the infection, and the anaerobic bacteria occur sporadically. The omission of bacteriological sample taking during surgery frustrates the targeted antibiotic treatment. The blood culturing may have useful contribution. The targeted antibiotic therapy based on relevant microbiological sample taking is substantial complementary of the surgical intervention in the treatment of the inflammatory pancreatic diseases.     

Two separate mechanisms of T cell clonal anergy to Mls-1

T cell tolerance to superantigen can be mediated by clonal anergy in which Ag-specific mature T cells are physically present but are not able to mount an immune response. We induced T cell unresponsiveness to minor lymphocyte stimulations locus antigen (Mls)-1a in mice transgenic for TCR V beta 8.1 in three different systems: 1) injection of Mls-1a spleen cells, 2) mating with Mls-1a mice, and 3) bone marrow (BM) chimeras in which Mls-1a is present only on nonhematopoietic cells. CD4+8-V beta 8.1+ cells from all these groups did not proliferate in response to irradiated spleen cells from Mls-1a mice. We compared the response of these cells by T cell/stimulator cell conjugate formation, Ca2+ mobilization, and proliferation assays. The mechanisms underlying the unresponsiveness of these T cells appear to differ. CD4+8-V beta 8.1+ cells from Mls-1a spleen cell-injected mice mobilized cytoplasmic Ca2+ but proliferated at a reduced level in response to cross-linking with anti-TCR mAb. However, these cells formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a when activated B cells were used as stimulators, although they produced reduced levels of IL-2. In Mls-1a/b V beta 8.1 transgenic mice, a subset in CD4+8-V beta 8.1+ cells did not mobilize cytoplasmic Ca2+ after TCR cross-linking. Their conjugate formation, Ca2+ mobilization, or proliferation in response to Mls-1a on activated B cells was undetectable. Finally, CD4+8-V beta 8.1+ cells from the BM chimeras proliferated to TCR cross-linking at a partially reduced level and formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a on activated B cells. These features suggest that the mechanisms underlying the maintenance of anergy in Mls-1a spleen cell-injected mice are distinct from those in Mls-1a mice.