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PubMed1996

Gestational cocaine and ethanol exposure alter spontaneous and cocaine-induced behavior in weanling rats.

The developmental and behavioral effects of prenatal exposure to cocaine and/or ethanol were examined in rats. Pregnant rats received ethanol (E; 2 g/kg, b.i.d.) orally, cocaine (C; 6 mg/kg/day, IV), or both (C/E) on gestational days 8-20. Controls consisted of pair-fed (PF) and untreated (UNT) groups. Offspring were weighed and examined for developmental markers beginning postnatal day one (PD1). On PD21 pups were individually observed in an open-field following either an injection of cocaine (10 mg/kg, IP), an injection of saline, or no treatment. Drug-treated and PF dams ate less food and gained less weight than the UNT dams. C and E litters had slightly increased mortality rates. Pups from both the C and E groups appeared less sensitive to the locomotor stimulant effect of cocaine. Pups from the E group engaged in significantly less spontaneous stereotypic locomotion than UNT and PF pups, while male pups from the C group exhibited a decrease in spontaneous exploratory behavior. Thus, prenatal exposure to C or E altered spontaneous and/or cocaine-induced behavior in weanling-aged rats, while the C/E combination did not augment either effect.

Animals↗
PubMed1997

Early development and developmental plasticity of the fasciculus gracilis in the North American opossum (Didelphis virginiana).

The first objective of the present study was to ask when axons of the fasciculus gracilis reach the nucleus gracilis in the North American opossum (Didelphis virginiana). When Fast Blue (FB) was injected into the lumbar cord on postnatal day (PD) 1 and the pups were killed 2 days later, labeled axons were present within a distinct fasciculus gracilis at thoracic and cervical levels of the cord. When comparable injections were made at PD3 or 5 and the pups were allowed to survive for the same time period, a few labeled axons could be followed to the caudal medulla where they were located dorsal to the presumptive nucleus gracilis. In order to verify these observations and to determine if any of the axons which innervate the nucleus gracilis early in development originate within dorsal root ganglia, we also employed cholera toxin conjugated to horseradish peroxidase (CT-HRP) to label dorsal root axons transganglionically. When CT-HRP was injected into the hindlimb on PD1 and the pups were maintained for 1 day prior to death and HRP histochemistry, labeled axons were present within the fasciculus gracilis at thoracic and cervical levels, but they could not be traced into the medulla. When comparable injections were made on PD3, and the pups were maintained for 2 days, labeled axons were present within the caudal medulla. Our second objective was to determine whether axons of the fasciculus gracilis grow through a lesion of their spinal pathway during early development. In one group of animals, the thoracic cord was transected at PD5, 8, 12, 20 and 26 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30-40 days later. After a 3-5 day survival, the pups were killed and perfused so that the spinal cord and brainstem could be removed and sectioned for fluorescence microscopy. In all of the cases lesioned at PD5, axons of the fasciculus gracilis were labeled rostral to the site of transection and they could be followed to the nucleus gracilis. Evidence for growth of fasciculus gracilis axons into the caudal medulla was also seen in cases lesioned at PD8. In contrast, labeled axons were not observed rostral to the lesion when it was made at PD12 or at later stages of development. In order to verify that some of the axons which crossed the lesion originated within dorsal root ganglia, the thoracic cord was transected at PD5 in another group of animals and 7 days later, injections of CT-HRP were made into one of the hindlimbs. After a 3 day survival, labeled axons could be traced through the lesion site and into the caudal medulla. We conclude that axons of the fasciculus gracilis reach the nucleus gracilis by at least PD5 in the opossum and that they grow through a lesion of their spinal pathway when it is made at the same age or shortly thereafter. The critical period for such growth appears to end between PD8 and PD12.

Amidines↗
PubMed1997

Alterations in the developing immune system of the F344 rat after perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin I. [correction of II]. Effects on the fetus and the neonate.

Perinatal exposure of rodents to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to result in thymic atrophy and cell-mediated immune suppression at lower doses than are required to produce those effects following adult exposure. This study was designed to examine the effects that in utero TCDD exposure has on thymocyte development in the rat. Timed-bred pregnant F344 rats were given 0, 1.0, or 3.0 mcg TCDD/kg body weight by gavage on gestational day 14 (GD14). On GD19 or GD22/postnatal day one (PD1), the dams were euthanized, and the dams and their offspring were examined for organ weight and thymus phenotypic alterations. GD19 fetuses from the 3.0 mcg TCDD/kg maternal exposure group exhibited decreases in relative thymus weight and thymic cellularity. There were a decreased percentage of CD3-/CD4+ CD8+ thymocytes and an increased percentage of CD3-/CD4-CD8+ thymocytes in these fetuses, but there were no alterations in the CD3+ subsets. No effects were seen in the GD19 fetuses from the 1.0 mcg TCDD/kg dosage group. In the TCDD-exposed GD22/PD1 offspring thymic atrophy was no longer present, but there was an increase in the relative liver weight. In addition, there were decreased percentages of CD3-/CD4-CD8-, CD3+/CD4-CD8-, and CD3+/CD4+CD8+ thymocytes and an increased percentage of CD3+/CD4-CD8+ thymocytes. The CD3+/CD4-CD8- and CD3+/CD4-CD8+ cell populations were the most sensitive, with changes appearing at both 1.0 and 3.0 mcg TCDD/kg maternal exposures. The TCDD-exposed GD19 dams exhibited an increased relative liver weight, a decreased relative thymus weight, and alterations in thymic CD3+ populations. Three days later the relative organ weights had recovered in the dams, but the phenotypic alterations were seen in CD3- as well as CD3+ thymocyte subsets. These results indicate that the developing rat fetal thymus is susceptible to the effects of TCDD. In addition, pregnant rats and their offspring showed similar alterations in thymocytic phenotypes.

Administration, Oral↗
PubMed1999

Gene expression of P2X-receptors in the developing inner ear of the rat.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to characterize the expression of P2X receptor subunits (P2X1-P2X7) in different inner ear tissues. The present study revealed the presence of P2X2, P2X3, P2X4 and P2X7-mRNA in rat organ of Corti, vestibular organ and spiral ganglion at different postnatal developmental stages (PD1-PD16), with slight differences in the onset of expression. Expression of P2X1, P2X5 and P2X6-mRNA was not detectable in the inner ear tissues. In addition, single cell RT-PCR experiments with outer hair cells (OHC) revealed the expression of either the P2X2 or the P2X2-2 splice variant or coexpression of both isoforms in individual cells. Our data suggest that extracellular adenosine-5'-triphosphate (ATP) may play an important role in signal transduction in the inner ear.

Aging↗
PubMed2002

Ontogeny of calmodulin gene expression in rat brain.

Calmodulin (CaM), a multifunctional intracellular calcium receptor, is a key element in signaling mechanisms. It is encoded in vertebrates by multiple apparently redundant genes (CaM I, II, III). To investigate whether differential expression takes place in the developing rat brain, a quantitative in situ hybridization analysis was carried out involving 15 brain areas at six ages between embryonic day 19 and postnatal day 20 (PD20) with gene-specific [(35)S]cRNA probes. A widespread, developmental stage-specific and differential expression of the three CaM genes was observed. The characteristic changes in the CaM mRNA levels in the examined time frame allowed the brain regions to be classified into three categories. For the majority of the areas (e.g. the piriform cortex for CaM III), the signal intensities peaked at around PD10 and the expression profile was symmetric (type 1). Other regions (e.g. the cerebral cortex, layer 1 for CaM II) displayed their highest signal intensities at the earliest age measured, followed by a gradual decrease (type 2). The signal intensities in the regions in the third group (e.g. the hypothalamus for CaM III) fluctuated from age to age (type 3). Marked CaM mRNA levels were measured for each transcript corresponding to the three CaM genes in the molecular layers of the cerebral and cerebellar cortici and hippocampus, suggesting their dendritic translocation. The highest signal intensity was measured for CaM II mRNA, followed by those for CaM III and CaM I mRNAs on PD1. However, the CaM II and CaM III mRNAs subsequently decreased steeply, while the CaM I mRNAs were readily detected even on PD20. Our results suggest that during development (1) the transcription of the CaM genes is under differential, area-specific control, and (2) a large population of CaM mRNAs is targeted to the dendritic compartment in a gene-specific manner.

Aging↗
PubMed2005

Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands.

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lck SH3 ligands using phage display. In addition, we analyzed Lck SH3 binding sites within known natural Lck SH3 binding proteins using an Lck specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lck SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lck SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands-which are not due to significant K(D) value differences as shown by calculating Lck SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lck SH3 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions.

Amino Acid Sequence↗
PubMed1999

Development of receptoral responses in pigmented and albino guinea-pigs (Cavia porcellus).

We describe the postnatal development of the electroretinogram (ERG) receptoral response in the guinea pig. In addition, the time course and nature of maturation was compared between albino and pigmented strains to consider the role that melanogenesis might have in this process. Electroretinograms were collected on groups of albino and pigmented animals from postnatal day (PD) PD1 to PD60. A-wave amplitudes and implicit times were extracted from filtered data (0-75 Hz). Receptoral components were modelled using the delayed gaussian model of Hood and Birch [1] fitted as an ensemble to the raw data. Guinea pigs show saturated amplitudes (RmP3) that are 50% of adult values at birth, these mature by PD12. Receptoral delay (t(d)) also undergoes some postnatal maturation, while phototransduction gain (log S) is adult-like at birth. Albino animals had significantly (p<0.05) larger RmP3 and log S across all ages. Guinea pigs have significant postnatal development in their receptoral response. Maturation of RmP3 implies a postnatal increase in rod outer segment length. Whereas the adult values of log S implies a mature phototransduction process at birth. We argue that the likely cause for the larger log S of albino eyes is compatible with theories of increased levels of internal light. Whereas the larger RmP3, even after allowing for increased light effectiveness, may reflect a lower ocular resistance in albino eyes due to their lower levels of melanin. Furthermore, decreased RmP3 and log S with age is observed in the pigmented group only and is consistent with increased ocular resistance due to melanin development in this strain.

Albinism, Oculocutaneous↗
PubMed2000

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein, D1.

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.

Amino Acid Substitution↗
PubMed2001

Evaluating pharmacokinetic/pharmacodynamic models using the posterior predictive check.

The posterior predictive check (PPC) is a model evaluation tool. It assigns a value (pPPC) to the probability that the value of a given statistic computed from data arising under an analysis model is as or more extreme than the value computed from the real data themselves. If this probability is too small, the analysis model is regarded as invalid for the given statistic. Properties of the PPC for pharmacokinetic (PK) and pharmacodynamic (PD) model evaluation are examined herein for a particularly simple simulation setting: extensive sampling of a single individual's data arising from simple PK/PD and error models. To test the performance characteristics of the PPC, repeatedly, "real" data are simulated and for a variety of statistics, the PPC is applied to an analysis model, which may (null hypothesis) or may not (alternative hypothesis) be identical to the simulation model. Five models are used here: (PK1) mono-exponential with proportional error, (PK2) biexponential with proportional error, (PK2 epsilon) biexponential with additive error, (PD1) Emax model with additive error under the logit transform, and (PD2) sigmoid Emax model with additive error under the logit transform. Six simulation/analysis settings are studied. The first three, (PK1/PK1), (PK2/PK2), and (PD1/PD1) evaluate whether the PPC has appropriate type-I error level, whereas the second three (PK2/PK1), (PK2 epsilon/PK2), and (PD2/PD1) evaluate whether the PPC has adequate power. For a set of 100 data sets simulated/analyzed under each model pair according to a stipulated extensive sampling design, the pPPC is computed for a number of statistics in three different ways (each way uses a different approximation to the posterior distribution on the model parameters). We find that in general; (i) The PPC is conservative under the null in the sense that for many statistics, prob(pPPC < or = alpha) < alpha for small alpha. With respect to such statistics, this means that useful models will rarely be regarded incorrectly as invalid. A high correlation of a statistic with the parameter estimates obtained from the same data used to compute the statistic (a measure of statistical "sufficiency") tends to identify the most conservative statistics. (ii) Power is not very great, at least for the alternative models we tested, and it is especially poor with "statistics" that are in part a function of parameters as well as data. Although there is a tendency for nonsufficient statistics (as we have measured this) to have greater power, this is by no means an infallible diagnostic. (iii) No clear advantage for one or another method of approximating the posterior distribution on model parameters is found.

Area Under Curve↗
PubMed2005

PDCD1: a tissue-specific susceptibility locus for inherited inflammatory disorders.

Variation in genes encoding costimulatory molecules expressed on lymphocytes has been expected to contribute to the genetic component of inflammatory disease, but only the gene encoding the inhibitory protein, CTLA-4, seems consistently to confer disease susceptibility. Studies in murine models implicate the inhibitory product of the pd1 gene, programmed death-1, in the maintenance of peripheral tolerance to self-antigens. We identify 22 single-nucleotide polymorphisms (SNPs) in the equivalent human gene, PDCD1, a number of which show significant associations with the specific immunoglobulin E response to grass allergens in atopic individuals. Stepwise analyses indicate that four of the disease-associated SNPs have independent effects. The two most common haplotypes show positive and negative associations but rarer haplotypes are also likely to be of influence. In a case-control study, multiple regression analysis of genotypic data implies that PDCD1 also confers susceptibility to rheumatoid arthritis. Along with work linking PDCD1 with susceptibility to another autoimmune condition, systemic lupus erythematosus, our data identify PDCD1 as a second immunomodulatory gene with pleiotropic effects in human disease. Genes encoding negative regulators may generally confer a significant fraction of the genetic risk associated with inherited inflammatory disorders.

Alleles↗
PubMed2005

A role of the C-terminal extension of the photosystem II D1 protein in sensitivity of the cyanobacterium Synechocystis PCC 6803 to photoinhibition.

The D1 protein, a key protein subunit of Photosystem II complex (PSII), is synthesised as a precursor (pD1) with a carboxyl-terminal extension. In the cyanobacterium Synechocystis sp. PCC 6803, this extension consists of 16 amino acid residues and it is cleaved by a specific protease in two putative steps with the final cleavage after the residue Ala344. In order to define the importance of the extension for the functioning of PSII, we constructed and characterized several site-directed mutants of Synechocystis that differ in the length and amino acid sequence of this extension. The mutant lacking the entire C-terminal extension exhibited slightly increased sensitivity to photoinhibition. Analysis of the PSII assembly in the mutant by the blue-native electrophoresis in combination with radioactive labelling revealed an increased level of the unassembled D1 protein in this strain. Replacement of the amino acid residue Asn359 by His or Asp also led to the higher vulnerability to photoinhibition of both mutants. In the Asn359His mutant, this vulnerability was accompanied by an increased level of the PSII core lacking CP43 indicating limitation of the repair cycle in the CP43 reassembly step.

Amino Acid Sequence↗
PubMed1998

Variable number of pig MHC class I genes in different serologically defined haplotypes identified by a 3'-untranslated region probe.

To estimate the number of porcine class I major histocompatibility genes, a short class I cDNA probe from the 3'-untranslated region was developed to be used in restriction fragment length polymorphism analysis. Six clones isolated from a pig spleen cDNA library were sequenced from their 3'-untranslated region. Three different transcripts were identified, one probably derived from the class I PD7 locus and two showing highest homology to the PD1 and the PD14 genes, respectively. Class I typing was performed both by restriction fragment length polymorphism and serology. Segregation of class I haplotypes was followed in one three-generation family (European Wild Boar x Large White: Swedish Yorkshire) and in six two-generation families (Duroc, Yorkshire and Chester White), for a total of 266 pigs. Twenty different class I haplotypes were identified either with restriction fragment length polymorphism and/or serological typing. Furthermore, previously unpublished serological haplotypes H62, H67 and H68 were identified. Two to seven polymorphic and three monomorphic fragments were detected in different restriction fragment length polymorphism haplotypes indicating that the number of class I genes in the investigated haplotypes varies.

Animals↗
PubMed2003

Jun oncoproteins do not function as primary transcription factors for the mouse major histocompatibility complex class I H-2 genes in fibroblasts.

There are several reports in the literature focusing on regulation of major histocompatibility complex (MHC) class I genes by transcription factors of the jun family. The methods employed in these reports differed in various respects, and their results are inconsistent. In mouse Lewis lung carcinoma, B16-melanoma and F9-teratocarcinoma cell lines, c-jun was characterized as a transcriptional activator of the murine MHC class I H2-Kb gene, while c-jun was identified as a direct transcriptional repressor of the swine class I PD1 gene, and c-jun stably transfected clones of mouse L-fibroblasts markedly reduced their H-2 class I gene expression. In this study, we attempted to reproduce this last effect by means of transient transfection coupled to Northern hybridization, upon transfecting L-fibroblasts with expression vectors for all jun family members as well as with an array of c-jun-derived dominant negative mutants. No change in H-2 class I expression could be identified. Next, we derived two additional fibroblastic cell lines from the fibrosarcoma of the H2-Kk/v-jun transgenic mouse and transfected them with the two most potent c-jun dominant negative mutants, again without eliciting any change in H-2 class I mRNA level. We conclude that the negative regulation of H-2 class I genes by c-jun in cells of the fibroblastic lineage is not a primary effect.

Animals↗
PubMed2002

Characterization, expression and functional aspects of a novel protein tyrosine phosphatase epsilon isoform.

This report describes the identification and characterization of a novel cytoplasmic isoform of human protein tyrosine phosphatase epsilon (PTPepsilon). The novel isoform, denoted cyt-PTPepsilonPD1, displays only the N-terminal catalytic, active phosphatase domain 1 (PD1) which is common in all known PTPepsilon isoforms. In addition, it contains a unique 132-residue long C-terminal end with no known motifs or homology to other characterized proteins. RNAse protection assay on isolated leucocyte subpopulations and selected cell lines demonstrated highest expression of cyt-PTPepsilonPD1 in monocytes. The mRNA-encoding cyt-PTPepsilonPD1 is detected as distinct transcript(s) by Northern blot analysis and is a result of alternative splicing. cyt-PTPepsilonPD1 shows similar cellular localization in transfected cells, both in the cytoplasm and nucleus, as has been previously described for cytoplasmic PTPepsilon isoform. Our previous data suggest that the expression of cytoplasmic PTPepsilon inhibits the mitogen-activated protein kinase cascade through the extracellular signal-regulated kinase 1 and 2 pathway. A similar functional role is also presented here for cyt-PTPepsilonPD1, supporting our previous data suggesting that the catalytic first PD of PTPepsilon is responsible for this inhibition.

Alternative Splicing↗
PubMed2001

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha.

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.

Adenylyl Cyclases↗
PubMed2005

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.

Amino Acid Sequence↗
PubMed1999

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

Amino Acid Sequence↗
PubMed2005

Naive CD4+ cells from cord blood can generate competent Th effector cells.

BACKGROUND: Umbilical cord blood (UCB) cells have been increasingly used as a source of hematopoietic stem cells for allogeneic transplantation. Previous reports suggest that the low risk of graft-versus-host disease in patients that received cord blood cells seems related to the distinctive nature of cord blood T cells. METHODS: To analyze the maturation of CD4+CD45RA+ cord blood cells, we performed an in vitro differentiation assay to compare the generation of Th effector cells strictly from UCB and adult peripheral blood (APB) CD4+CD45RA+ cells. RESULTS: During the maturation into effector cells, UCB and APB cells acquired a comparable activation level determined by the expression pattern of CD69, CD40L, OX40 and CD62L as well as PD1 and CTLA-4 molecules. Moreover, the expression of CD45RO isoform was induced in most activated effector cells from both UCB and APB. OKT3-restimulated effector cells generated from naive UCB expressed higher levels of CD25 coinciding with the secretion of higher amounts of IL-2. Effector cells from both origins consisted of heterogeneous populations with similar frequencies of Th1 and Th2 cytokine producing cells, secreting equivalent levels of IL-4, IL-5 and IFNgamma. Although, higher levels of IL-10 were detected in the cytokine mRNA profile and in the supernatants of OKT3-restimulated UCB effector cells, blocking endogenous IL-10 with anti-IL-10 mAbs enhanced significantly the proliferative response of UCB as well as APB effector cells (P < 0.05). CONCLUSIONS: These results indicated that Th effector cells generated from naive UCB cells were intrinsically as competent as naive APB to respond to TCR-mediated stimulation. In addition, UCB effector cells produced higher IL-10 but its inhibitory effect on proliferation may be partially compensated by the higher production of IL-2 and enhanced expression of CD25.

Biomarkers↗