Purification and characterization of a hepatic mitochondrial cytochrome P-450 active in aflatoxin B1 metabolism

We have previously shown that phenobarbital (PB) increases hepatic mitochondrial cytochrome P-450 (P-450) content and also the ability to metabolize hepatocarcinogen, aflatoxin B1 [Niranjan, B. G., Wilson, N. M., Jefcoate, C. R., & Avadhani, N. G. (1984) J. Biol. Chem. 259, 12495-12501]. In the present study, we have purified a mitochondrial-specific P-450 with an apparent molecular mass of 52 kdaltons (termed P-450mt3) from PB-induced rat liver using a combination of hydrophobic and ion exchange column chromatography procedures. Polyclonal antibody to P-450mt3 failed to cross-react with P-450mt1 and P-450mt2 purified from beta-naphthoflavone- (BNF) induced rat liver mitochondria. Furthermore, P-450mt3 shows an N-terminal amino acid sequence (Ala-Ile-Pro-Ala-Ala-Leu-Arg-Thr-Asp) different from those of both P-450mt1 and P-450mt2, as well as microsomal P-450b. The polyclonal antibody to P-450mt3 cross-reacted with a P-450 of comparable size purified from uninduced mitochondria. These two isoforms, however, showed difference with respect to catalytic properties and amino acid composition. In vitro reconstitution experiments show that P-450mt3 can actively metabolize diverse substrates including (dimethylamino)antipyrine, benzphetamine, and aflatoxin B1 but shows a low vitamin D3 25-hydroxylase activity. The mitochondrial P-450 from uninduced livers, on the other hand, shows relatively high [229 pmol min-1 (nmol of P-450)-1] vitamin D3 25-hydroxylase activity but a considerably lower ability for aflatoxin B1 metabolism and no detectable activity for (dimethylamino)antipyrine and benzphetamine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

<Emphasis Type="Italic">Pichia stipitis</Emphasis> xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

Background  

Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.     

Surface structure changes of rat adipocytes during lypolysis stimulated by various lypolysis stimulated by various lypolytic agents

A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 μM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

Adding Content to Contacts: Measurement of High Quality Contacts for Maternal and Newborn Health in Ethiopia,North East Nigeria,and Uttar Pradesh,India

BackgroundFamilies in high mortality settings need regular contact with high quality services, but existing population-based measurements of contacts do not reflect quality. To address this, in 2012, we designed linked household and frontline worker surveys for Gombe State, Nigeria, Ethiopia, and Uttar Pradesh, India. Using reported frequency and content of contacts, we present a method for estimating the population level coverage of high quality contacts.ConclusionsMeasuring content of care to reflect the quality of contacts can reveal missed opportunities to deliver best possible health care.     

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.     

The upstream open reading frame mediates constitutive effects on translation of cytochrome p-450c27 from the seventh in-frame AUG codon in rat liver

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55-kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression.