Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben)

Studies were conducted to elucidate the importance of androgen-mediated induction of the extreme masculinization of the external genitalia in female spotted hyenas. Phallic size and shape; androgen receptor (AR) and alpha-actin expression; and sex-specific differences in phallic retractor musculature, erectile tissue, tunica albuginea, and urethra/urogenital sinus were examined in male and female fetuses from Day 30 of gestation to term. Similar outcomes were assessed in fetuses from dams treated with an AR blocker and a 5alpha-reductase inhibitor (antiandrogen treatment). Clitoral and penile development were already advanced at Day 30 of gestation and grossly indistinguishable between male and female fetuses throughout pregnancy. Sex-specific differences in internal phallic organization were evident at Gestational Day 45, coincident with AR expression and testicular differentiation. Antiandrogen treatment inhibited prostatic development in males and effectively feminized internal penile anatomy. We conclude that gross masculinization of phallic size and shape of male and female fetuses is androgen-independent, but that sexual dimorphism of internal phallic structure is dependent on fetal testicular androgens acting via AR in the relevant cells/tissues. Androgens secreted by the maternal ovaries and metabolized by the placenta do not appear to be involved in gross masculinization or in most of the sex differences in internal phallic structure.     

A 2500-locus bin map of wheat homoeologous group 5 provides insights on gene distribution and colinearity with rice

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).     

Variations in the human phospholipase Cγ2 gene in patients with B-cell defects of unknown etiology

Our recent studies using targeted gene disruption have shown that defects in phospholipase Cgamma2 (PLCgamma2) result in a B-cell abnormality that is very similar to that seen in Btk-deficient mice. Null mutations in either PLCG2 or BTK are associated with decreased numbers of mature B cells, failure to make antibodies to some T cell-independent antigens and the absence of CD5+ peritoneal B cells. Mutations in BTK in humans cause a more severe defect in B-cell development characterized by almost complete absence of B cells in the peripheral circulation, profound hypogammaglobulinemia and an inability to produce antibodies to any antigens. However, not all patients with severe defects in B-cell development have mutations in BTK or the components of the B-cell signal transduction complex. To explore the possibility that some patients with defects in B-cell development of unknown etiology might have mutations in PLCG2, we determined the genomic structure of this gene and established conditions to analyze the 32 exons of the gene and the flanking sequences by single-strand conformation polymorphism. Although 24 polymorphic variants of this gene were found in 35 patients, we did not identify any alterations that were likely to be the cause of disease.     

Differential Effects of AGS3 Expression on D2L Dopamine Receptor-Mediated Adenylyl Cyclase Signaling

Activator of G protein signaling 3 (AGS3) binds Gα_i subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα_i-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D_2L dopamine receptor (D_2LDR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D_2LDR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D_2LDR-mediated inhibition of AC1, whereas desensitization of D_2LDR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D_2LDR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.     

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.     

The ecological integrity of the lower Olifants River,Limpopo province,South Africa: 2009–2015 – Part B: Tributaries of the Olifants River

Monitoring on the Lowveld reaches of the Olifants River, Limpopo River System, and its Steelpoort, Blyde, Klaserie and Selati tributaries was initiated in 2009. Analysis of the 2009–2015 data from four Olifants River sites showed deterioration in the river’s ecological condition between where it enters the Lowveld and where it enters the Kruger National Park, with a slight recovery within the Kruger National Park. Physico-chemical, aquatic macroinvertebrate and fish data collected in 2009–2015 at six sites on the Steelpoort, Blyde, Klaserie and Selati tributaries of the Olifants River corroborated the ecological condition of these tributaries. The Selati was the most polluted and was in a critically modified condition, whereas the Klaserie and Steelpoort were in fair condition and the Blyde was in good condition. The Selati appeared to have a significant negative impact on the water quality, macroinvertebrates and fish of the Olifants River within the Kruger National Park.