Glial-specific ablation of angiotensinogen lowers arterial pressure in renin and angiotensinogen transgenic mice

Angiotensinogen (AGT) is mainly expressed in glial cells in close proximity to renin-expressing neurons in the brain. We previously reported that glial-specific overexpression of ANG II results in mild hypertension. Here, we tested the hypothesis that glial-derived AGT plays an important role in blood pressure regulation in hypertensive mice carrying human renin (hREN) and human AGT transgenes under the control of their own endogenous promoters. To perform a glial-specific deletion of AGT, we used an AGT transgene containing loxP sites (hAGT(flox)), so the gene can be permanently ablated in the presence of cre-recombinase expression, driven by the glial fibrillary acidic protein (GFAP) promoter. Triple transgenic mice (RAC) containing a: 1) systemically expressed hREN transgene, 2) systemically expressed hAGT(flox) transgene, and 3) GFAP-cre-recombinase were generated and compared with double transgenic mice (RA) lacking cre-recombinase. Liver and kidney hAGT mRNA levels were unaltered in RAC and RA mice, as was the level of hAGT in the systemic circulation, consistent with the absence of cre-recombinase expression in those tissues. Whereas hAGT mRNA was present in the brain of RA mice (lacking cre-recombinase), it was absent from the brain of RAC mice expressing cre-recombinase, confirming brain-specific elimination of AGT. Immunohistochemistry revealed a loss of AGT immunostaining glial cells throughout the brain in RAC mice. Arterial pressure measured by radiotelemetry was significantly lower in RAC than RA mice and unchanged from nontransgenic control mice. These data suggest that there is a major contribution of glial-AGT to the hypertensive state in mice carrying systemically expressed hREN and hAGT genes and confirm the importance of a glial source of ANG II substrate in the brain.     

The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria

We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.     

Empirical choice of sampling procedures for optimal research design in the longitudinal study of primate behavior

A method is suggested to evaluate on an empirical basis sampling plans for the longitudinal study of primate behavior in those very common situations in which the mathematical structure of behavior is unknown. The method is based on a randomization procedure applied to a pilot sample of the behavior organized so that cost of implementation of a sampling plan can be evaluated vis á vis the sampling error intrinsic to the plan.     

Unprecedented 34S‐enrichment of pyrite formed following microbial sulfate reduction in fractured crystalline rocks

In the deep biosphere, microbial sulfate reduction (MSR) is exploited for energy. Here, we show that, in fractured continental crystalline bedrock in three areas in Sweden, this process produced sulfide that reacted with iron to form pyrite extremely enriched in ³⁴S relative to ³²S. As documented by secondary ion mass spectrometry (SIMS) microanalyses, the δ³⁴S_pyrite values are up to +132‰V‐CDT and with a total range of 186‰. The lightest δ³⁴S_pyrite values (?54‰) suggest very large fractionation during MSR from an initial sulfate with δ³⁴S values (δ³⁴S_sulfate,0) of +14 to +28‰. Fractionation of this magnitude requires a slow MSR rate, a feature we attribute to nutrient and electron donor shortage as well as initial sulfate abundance. The superheavy δ³⁴S_pyrite values were produced by Rayleigh fractionation effects in a diminishing sulfate pool. Large volumes of pyrite with superheavy values (+120 ± 15‰) within single fracture intercepts in the boreholes, associated heavy average values up to +75‰ and heavy minimum δ³⁴S_pyrite values, suggest isolation of significant amounts of isotopically light sulfide in other parts of the fracture system. Large fracture‐specific δ³⁴S_pyrite variability and overall average δ³⁴S_pyrite values (+11 to +16‰) lower than the anticipated δ³⁴S_sulfate,0 support this hypothesis. The superheavy pyrite found locally in the borehole intercepts thus represents a late stage in a much larger fracture system undergoing Rayleigh fractionation. Microscale Rb–Sr dating and U/Th–He dating of cogenetic minerals reveal that most pyrite formed in the early Paleozoic era, but crystal overgrowths may be significantly younger. The δ¹³C values in cogenetic calcite suggest that the superheavy δ³⁴S_pyrite values are related to organotrophic MSR, in contrast to findings from marine sediments where superheavy pyrite has been proposed to be linked to anaerobic oxidation of methane. The findings provide new insights into MSR‐related S‐isotope systematics, particularly regarding formation of large fractions of ³⁴S‐rich pyrite.     

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.     

Non-imidazole heterocyclic histamine H3 receptor antagonists

Continued exploration of the SAR around the lead imidazopyridine histamine H(3) antagonist 1 has led to the discovery of several related series of heterocyclic histamine H(3) antagonists. The synthesis and SAR of indolizine, indole and pyrazolopyridine based compounds are now described.     

Stimulation of Pod and Ovule Growth of Soybean, Glycine max (L.) Merr. by 6-Benzylaminopurine

Flowers at distal nodes on soybean racemes usually fail to setpods and subsequently abscise. Physiological and histologicalstudies were performed to determine the influence of 6-benzylaminopurine(BAP) on distal pod development. The pedicels of fully openedflowers on terminal racemes of field-grown IX93-100 soybeanplants were treated three times with 200 mg kg^-1 BAP in lanolinover a 6-d period. Racemes were then excised and ³²P uptakewas recorded for each flower position within a raceme; histologicalfeatures of pedicels and ovules also were determined. Applicationof BAP increased pod and ovule length, width and weight at allfour distal nodes (D, D-1, D-2, D-3) relative to controls treatedwith lanolin. Length and width of parietal endosperm cells weresmaller in BAP-treated ovules at the most proximal node beingstudied (D-3), and greater numbers of parietal endosperm cellswere observed at D-1 and D-3 nodes when compared to lanolincontrols. Smaller amounts of starch were found in suspensorcells, endosperm, and integuments of lanolin-treated ovules,and starch depletion over time was observed within starch sheathsof pedicels from lanolin-treated pods when compared to BAP-treatedtissues. BAP-treated racemes had more ³²P uptake at the fourmost distal nodes. A higher rate of uptake (cpm mg^-1 f. wt)was evident in ovules than in ovary tissues. These results suggestthat for racemes otherwise destined to abscise, applicationof BAP promotes pod set and growth by stimulating ovule development.Copyright1993, 1999 Academic Press Pod, ovule soybean, abscission, 6-benzylaminopurine     

Novel tetrahydropyrido[3,2-c]pyrroles as 5-HT(7) antagonists

The synthesis and SAR for a novel series of tetrahydropyrido[3,2-c]pyrroles is described. Optimization of the pendant aryl ring lead to high binding affinity at the 5-HT₇ receptor when small lipophilic groups were placed in the para position. Modification of the N-benzyl group and secondary amine were not well tolerated. A representative set of compounds was shown to be functional antagonists of the 5-HT₇ receptor.