Treatment with non-hypercalcemic analogs of 1,25-dihydroxyvitamin D3 increases responsiveness to 17beta-estradiol, dihydrotestosterone or raloxifene in primary human osteoblasts

Pretreatment with 1 nM 1,25-dihydroxyvitamin D(3) (1,25), or non-hypercalcemic Vitamin D analogs, upregulated the response of creatine kinase (CK) to 17beta-estradiol (30 nM E(2)), raloxifene (3000 nM RAL) or dihydrotestosterone (300 nM DHT) in primary human bone cells. Previously, we reported that these osteoblast-like cells responded to gonadal steroids in a sex specific manner. Bone cells derived from pre-menopausal women showed greater stimulation of CK specific activity by E(2) than bone cells from post-menopausal women; in male-derived cells no age related difference was found. In this study, we treated cells derived from female or male bones, at different ages, with the side chain modified analogs of Vitamin D: CB 1093 (CB), EB 1089 (EB), MC 1288 (MC) and the demonstrably non-calcemic hybrid analog JK 1624 F2-2 (JKF), by daily addition of 1 nM, for 3 days. On day 4, cells were incubated with sex steroids for 4h and cell extracts were prepared. Pretreatment with JKF or CB significantly upregulated the response to 30 nM E(2) in all female-derived cells and to 300 nM DHT in mature male-derived cells. In cells from older males, only JKF caused augmentation of DHT action. Bone cells from pre- or post-menopausal females responded to 3000 nM RAL by increased CK activity to the same extent as to 30 nM E(2); however, RAL and E(2), when applied together, resulted in mutual annihilation of their agonist activities. Vitamin D analogs prevented the antagonistic effect of RAL in the presence of E(2), possibly due to increased numbers of ERs. Both estrogen receptors, alpha (ERalpha) and beta (ERbeta), were expressed in male- as well as in female-derived cells. However, only in female-derived cells were ERalpha and ERbeta upregulated by pretreatment with Vitamin D analogs. This study raises the possibility of testing combined Vitamin D analog and estrogen replacement treatment for post-menopausal women to prevent osteoporosis.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Allosteric interactions among pyrethroid,brevetoxin, and scorpion toxin receptors on insect sodium channels raise an alternative approach for insect control

Intensive pyrethroid use in insect control has led to resistance buildup among various pests. One alternative to battle this problem envisions the combined use of synergistically acting insecticidal compounds. Pyrethroids, scorpion - and β-toxins, and brevetoxins bind to distinct receptor sites on voltage-gated sodium channels (NaChs) and modify their function. The binding affinity of scorpion -toxins to locust, but not rat-brain NaChs, is allosterically increased by pyrethroids and by brevetoxin-1. Brevetoxin-1 also increases the binding of an excitatory β-toxin to insect NaChs. These results reveal differences between insect and mammalian NaChs and may be exploited in new strategies of insect control.     

The palaeoecology of the Upper Ndolanya Beds at Laetoli,Tanzania

The palaeoecology of the fauna from the Ndolanya Beds, Laetoli, Tanzania, has been analysed to reconstruct the environment of this 2.6 Ma site. Community profiles have been constructed in relation to three variables that carry ecological meaning: body weight, locomotor adaptations and feeding preferences. Comparing the similarities and differences in the structure of the fossil faunas with those of modern environments allows us to draw inferences about palaeoenvironmental conditions, and this is based on a comparative dataset of 15 modern environments (44 localities) covering a wide range of climatic and ecological conditions across Asia, Africa and Central America. In addition, 16 fossil sites in East and South Africa have been analysed in the same way, and both sets of comparative data have been used as the basis for comparison with an ecological diversity analysis of the fauna from the Upper Ndolanya Beds. The Ndolanya fauna is characterized by a predominance of medium to large sized terrestrial and herbivorous species.There is evidence of taphonomic bias that eliminated many of the smaller species. A comparison of multivariate analyses of 23 selected modern localities conducted both with and without the small species included, indicates that the loss of these species does not adversely influence the results of an ecological diversity analysis. The evidence suggests that at the time of the deposition of the Ndolanya Beds the Laetoli region was a semi-arid bushland. This is considerably drier and more open than is suggested for the earlier Laetolil Beds.     

The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid-base catalytic residue. AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.     

Diazoxide and Pinacidil Uncouple Pyruvate–Malate-Induced Mitochondrial Respiration

We investigated the effects of K_ATP channel openers diazoxide and pinacidil on the respiration rate and membrane potential () of rat heart mitochondria, oxidizing pyruvate and malate. Diazoxide and pinacidil (58.8–1348.3 M) increased the V ₂ (-ADP) respiration rate accordingly by 13–208% and 30–273% and decreased the by 2–17% and 6–55%. These effects were also similar in the respiration medium without K⁺. Moreover, carboxyatractyloside completely abolished diazoxide- and pinacidil-induced uncoupling, indicating a role for the mitochondrial adenine nucleotide translocase in this process.     

Aplidiasterols A and B, two new cytotoxic 9,11-secosterols from the Mediterranean ascidian Aplidium conicum

Two novel 9,11-secosterols, aplidiasterols A (3β,6β,11-trihydroxy-9,11-seco-5α-cholest-7-en-9-one, 1) and B (3β,5α,6β,11-tetrahydroxy-9,11-secocholest-7-en-9-one, 2), along with the known secosterols 3 and 4, were isolated from the Mediterranean ascidian Aplidium conicum and their structures were determined by spectroscopic data. Aplidiasterols A and B were found to be cytotoxic against rat glioma (C6) and murine monocyte/macrophage (J774) tumor cells in vitro. Compounds 1-4 represent the first example of secosterols isolated from tunicates.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Tumor necrosis factors in a murine model of allergic peritonitis: effects on eosinophil accumulation and inflammatory mediators' release

In allergic disorders, the role of tumor necrosis factors (TNF) is not well established. We investigated the role of TNF in allergic peritonitis induced by ovalbumin (OVA) challenge in double TNF (TNF-alpha(-/-)/lymphotoxin-alpha(-/-)) knock out (TNF-KO) mice. In the peritoneal lavage of TNF-KO mice, mast cell number and histamine level (radioenzymatic assay) were similar to that in wild type (WT) mice. TNF-alpha (ELISA) and histamine were increased 1 h after challenge in WT mice. However, three days later eosinophil number and eosinophil peroxidase (EPO) levels (colorimetric-enzymatic assay) were found to be lower in TNF-KO mice. A second challenge three days after the first, increased EPO, histamine and IL-6 (ELISA) but did not alter eosinophil and mast cell numbers in both types of mice. On the other hand histamine and IL-6 were higher, while EPO was lower in TNF-KO mice. In conclusion, our findings show that TNF is involved in eosinophil accumulation and inflammatory mediators' release in a murine model of allergy.     

Masseter muscle activity during vestibular stimulation in man

Experimental data report that vestibular afferents affect trigeminal system activity. The aim of this work was to evaluate whether static vestibular stimulation affects the excitability of trigeminal motoneurons in man. In order to assess this, voluntary EMG activity of masseter muscles as well as duration and latency of the early and late components of EMG exteroceptive silent period were evaluated while keeping the subject in vertical position and during 20 degrees static tilt. The experiments were performed on ten adult subjects with no orofacial, neurologic and otologic disorders. Each subject sat on a chair, which kept the complex head-jaw-neck-trunk and the limbs securely fixed, in order to minimize any interference due to the activation of somatosensory and proprioceptive afferents from these districts. The subjects were instructed to contract masseter muscles at 25% of their maximum bite force and the isometric force monitoring was used as visual feedback. Exteroceptive silent period (ESP) of masseter EMG was elicited by electrically stimulating the inferior inter-incisal gum. Results showed that static vestibular stimulation induced asymmetrical responses on voluntary masseter muscle activity, which was reduced to 70.3 +/- 16.1% (mean +/- S.D.) of the control value during ipsilateral tilt and increased to 128.8 +/- 13.0% during contralateral tilt. The duration of the early (ESP1) and late (ESP2) silent periods was also affected: during ipsilateral tilt ESP1 and ESP2 duration increased to 130.0 +/- 3.5% and to 122.1 +/- 2.1% of control, respectively; during contralateral tilt it was reduced to 76.8 +/- 1.2% and to 83.0 +/- 1.7% of control, respectively. On the contrary, changes in latencies were not significant. These data evidenced an asymmetrical effect exerted on trigeminal motor activity by static tilt. Since the influence of all receptors which could be activated by static tilt, except that arising from the macular ones, was minimized in this study, it is likely that the observed effects, induced by static tilt on masseter muscle activity, were of macular origin.