Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Cryotrapped reaction intermediates of cytochrome p450 studied by radiolytic reduction with phosphorus-32

Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using (60)Co gamma-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe(3+)-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999) J. Am. Chem. Soc. 121, 10654-10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at approximately 195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed.     

Primary Care Physicians’ Attitudes about Obesity and Its Treatment

Objective: This study was designed to assess physicians’ attitudes toward obese patients and the causes and treatment of obesity. Research Methods and Procedures: A questionnaire assessed attitudes in 2 geographically representative national random samples of 5000 primary care physicians. In one sample (N = 2500), obesity was defined as a BMI of 30 to 40 kg/m², and in the other (N = 2500), obesity was defined as a BMI > 40. Results: Six hundred twenty physicians responded. They rated physical inactivity as significantly more important than any other cause of obesity (p < 0.0009). Two other behavioral factors—overeating and a high‐fat diet—received the next highest mean ratings. More than 50% of physicians viewed obese patients as awkward, unattractive, ugly, and noncompliant. The treatment of obesity was rated as significantly less effective (p < 0.001) than therapies for 9 of 10 chronic conditions. Most respondents (75%), however, agreed with the consensus recommendations that a 10% reduction in weight is sufficient to improve obesity‐related health complications and viewed a 14% weight loss (i.e., 78 ± 5 kg from an initial weight of 91 kg) as an acceptable treatment outcome. More than one‐half (54%) would spend more time working on weight management issues if their time was reimbursed appropriately. Discussion: Primary care physicians view obesity as largely a behavioral problem and share our broader society's negative stereotypes about the personal attributes of obese persons. Practitioners are realistic about treatment outcomes but view obesity treatment as less effective than treatment of most other chronic conditions.     

Pepino mosaic virus triple gene block protein 1 (TGBp1) interacts with and increases tomato catalase 1 activity to enhance virus accumulation

Various plant factors are co‐opted by virus elements (RNA, proteins) and have been shown to act in pathways affecting virus accumulation and plant defence. Here, an interaction between Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1; p26) and tomato catalase 1 (CAT1), a crucial enzyme in the decomposition of toxic hydrogen peroxide (H₂O₂), was identified using the yeast two‐hybrid assay, and confirmed via an in vitro pull‐down assay and bimolecular fluorescent complementation (BiFC) in planta. Each protein was independently localized within loci in the cytoplasm and nuclei, sites at which their interaction had been visualized by BiFC. Following PepMV inoculation, CAT mRNA and protein levels in leaves were unaltered at 0, 3 and 6 days (locally) and 8 days (systemically) post‐inoculation; however, leaf extracts from the last two time points contained increased CAT activity and lower H₂O₂ levels. Overexpression of PepMV p26 in vitro and in planta conferred the same effect, suggesting an additional involvement of TGBp1 in potexvirus pathogenesis. The accumulation of PepMV genomic and subgenomic RNAs and the expression of viral coat protein in noninoculated (systemic) leaves were reduced significantly in CAT‐silenced plants. It is postulated that, during PepMV infection, a p26–CAT1 interaction increases H₂O₂ scavenging, thus acting as a negative regulator of plant defence mechanisms to promote PepMV infections.     

Does Vitamin D affects changes in volumetric bone mineral density and architecture in postmenopausal women after conservatively treated distal radius fractures?

Objective:We examined the role of vitamin D on volumetric bone mineral density (vBMD) and architecture during the first week’s post-fracture in postmenopausal women (PMW) with distal radial fractures (DRF) treated conservatively using peripheral Quantitative Computed Tomography (pQCT).Methods:Patients were classified into 2 groups according to initial median 25(OH)D level; Group A (25(OH)D ≥15 ng/ml) and group B (25(OH)D <15 ng/ml). All patients were followed for 12 weeks at three visits: baseline, 6 weeks and 12 weeks post fracture. pQCT was performed at baseline in fractured and contralateral non-fractured radius and at 6^th and 12^th week on the fractured side.Results:39 patients completed the protocol. Mean 25(OH)D levels were 15.60±7.35 ng/ml (3.5-41.7). Trabecular (trab) bone mineral content (BMC) and trabvBMD increased at 6 wk. vs. baseline (p<0.001). Cortical BMC, cortvBMD and cross- sectional area (CSA) progressively decreased (p<0.001) during the 12 weeks. There was no interaction between baseline 25(OH)D levels and changes in trabecular and cortical BMC, vBMD and CSA. Advanced age and higher CTX and P1NP were associated with higher cortical bone loss.Conclusion:Vitamin D deficiency does not affect the early architectural changes after a DRF. Advanced age and higher bone remodeling were associated with higher cortical bone loss, probably related to immobilization and independent of vitamin D levels.     

Tumor necrosis factor α induces a model of preeclampsia in pregnant baboons (Papio hamadryas)

Preeclampsia is a common disease of pregnancy characterised by maternal hypertension and proteinuria. Abnormal placentation in early pregnancy and abnormal cytokine and anti-angiogenic factor expression are thought to contribute to the clinical syndrome of endothelial dysfunction evident in the second half of gestation. The mechanisms underlying both the placental pathology and its translation to the maternal clinical syndrome are not fully understood. A model of preeclampsia manifest by clinically evident endothelial dysfunction (increased blood pressure and proteinuria) was induced by administration of low-dose TNF-α for 2 weeks at mid-gestation in pregnant baboons (Papio hamadryas). Blood pressure was monitored continuously and remotely by intra-arterial radiotelemetry. Following TNF-α infusion, there was an increase in systolic and diastolic blood pressure and development of proteinuria in pregnant treated animals, but not in pregnant saline controls nor in non-pregnant TNF-α treated animals. The treated pregnant animals also developed elevated plasma soluble FMS-like tyrosine kinase-1 (sFLT-1) and increased placental mRNA expression of sFLT-1 and soluble endoglin (sEng). These results clearly demonstrate that the cytokine TNF-α can induce the clinical and biochemical features of human preeclampsia. The results identify a link between cytokines, placental dysfunction and endothelial dysfunction resulting in a loss of maternal blood pressure control.     

The study of the E-class SEPALLATA3-like MADS-box genes in wild-type and mutant flowers of cultivated saffron crocus (Crocus sativus L.) and its putative progenitors

To further understand flowering and flower organ formation in the monocot crop saffron crocus (Crocus sativus L.), we cloned four MIKC^c type II MADS-box cDNA sequences of the E-class SEPALLATA3 (SEP3) subfamily designated CsatSEP3a/b/c/c_as as well as the three respective genomic sequences. Sequence analysis showed that cDNA sequences of CsatSEP3 c and c_as are the products of alternative splicing of the CsatSEP3c gene. Bioinformatics analysis with putative orthologous sequences from various plant species suggested that all four cDNA sequences encode for SEP3-like proteins with characteristic motifs and amino acids, and highlighted intriguing sequence features. Phylogenetically, the isolated sequences were closest to the SEP3-like genes from monocots such as Asparagus virgatus, Oryza sativa, Zea mays, and the dicot Arabidopsis SEP3 gene. All four isolated C. sativus sequences were strongly expressed in flowers and in all flower organs: whorl1 tepals, whorl2 tepals, stamens and carpels, but not in leaves. Expression of CsatSEP3a/b/c/c_as cDNAs was compared in wild-type and mutant flowers. Expression of the isolatedCsatSEP3-like genes in whorl1 tepals together with E-class CsatAP1/FUL subfamily and B-class CsatAP3 and CsatPI subfamilies of genes, fits the ABCE “quartet model,” an extended form of the original ABC model proposed to explain the homeotic transformation of whorl1 sepals into whorl1 tepals in Liliales and Asparagales plants such as C. sativus. This conclusion was also supported by the interaction of the CsatSEP3b protein with CsatAP1/FUL and CsatAP3 proteins. In contrast, expression of both B-class CsatAP3 and CsatPI genes and the C-class CsatAGAMOUS genes together with E-class CsatSEP3-like genes in carpels, without any phenotypic effects on carpels, raises questions about the role of these gene classes in carpel formation in this non-grass monocot and requires further experimentation. Finally, taking advantage of the size and sequence differences in amplified genomic sequences of the triploid C. sativus and comparing them with the respective sequences from C. tomasii, C. hadriaticus and C. cartwrightianus, three putative wild-type diploid progenitor species, we examined the origin of CsatSEP3a sequence.