Degradation of Dehydrodivanillin by Anaerobic Bacteria from Cow Rumen Fluid

Dehydrodivanillin (DDV; 0.15 g/liter) was biodegradable at 37°C under strictly anaerobic conditions by microflora from cow rumen fluid to the extent of 25% within 2 days in a yeast extract medium. The anaerobes were acclimated on DDV for 2 weeks, leading to DDV-degrading microflora with rates of degradation eight times higher than those initially. Dehydrodivanillic acid and vanillic acid were detected in an ethylacetate extract of a DDV-enriched culture broth by thin-layer, gas, and high-performance liquid chromatographies and by mass spectrometry.     

Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.     

Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: species differences between rat and mouse

Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells.     

Regulation of luteal cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by estradiol

Recent studies have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular sterol availability and metabolism. This investigation was performed to examine the effect of estradiol on de novo synthesis of cholesterol. Pregnant rats hypophysectomized and hysterectomized on Day 12 were treated for 72 h with either estradiol or testosterone. De novo cholesterol synthesis was determined by measurement of the specific activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, in microsome-enriched preparations of luteal tissue and incorporation of [14C] acetate into cholesterol by corpora lutea incubated in vitro. Estradiol or testosterone treatment caused a 4- to 5-fold stimulation of luteal cholesterol biosynthesis, as measured by these techniques. NaF, an inhibitor of phosphatase which blocks the conversion of the inactive enzyme to the active form, reduced the HMG CoA reductase activity to 30% in corpora lutea obtained from either steroid or vehicle-treated rats. However, an increase in enzyme activity of comparable magnitude by steroids was observed whether microsomes were isolated with or without NaF. The effect of estradiol appears to be enzyme-specific, since it failed to affect the microsomal marker, NADPH-cytochrome c reductase. Since the cholesteryl ester content of corpora lutea falls in response to steroid treatment, rats were treated with 4-aminopyrazolo-[3,4d]pyrimidine (4-APP) to deplete cellular cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental study of physiological pulsatile flow past valve prostheses in a model of human aorta--I. Caged ball valves

Pulsatile flow development past a caged ball valve in a model human aorta was studied using laser Doppler anemometry. Velocity profiles measured in the ascending aorta and in the mid-arch region were strongly influenced by the geometry of the valve at the root of the aorta. Velocity profiles distal to the valve were asymmetric with jet-like flow in the peripheral region having larger velocity magnitudes towards the left lateral wall. In early diastole, a streamwise vortex motion was observed throughout the model aorta with fluid moving towards the downstream direction along the left lateral wall and reversed flow along the right lateral wall. With the caged ball valve at the root of the aorta, no reversed flow was observed along the inner wall of curvature in the mid-arch region.     

Effect of different phospholipids on the reconstitution of two functions of the lactose carrier ofEscherichia coli

Summary The lactose carrier was extracted from membranes ofEscherichia coli and transport activity reconstituted in proteoliposomes containing different phospholipids. Two different assays f for carrier activity were utilized: counterflow and membrane potential-driven uptake. Proteoliposomes composed ofE. coli lipid or of 50% phosphatidylethanolamine–50% phosphatidylcholine showed very high transport activity with both assays. On the other hand, proteoliposomes containing asolectin, phosphatilcholine or 25% cholesterol/75% phosphatidylcholine showed good counterflow activity but poor membrane potentialdriven uptake. The discrepancy between the two types of transport activity in the latter group of three lipids is not due to leakiness to protons, size of proteoliposomes, or carrier protein content per proteoliposome. Apparently one function of the carrier molecule shows a broad tolerance for various phospholipids, while a second facet of the membrane protein activity requires very restricted lipid enviroment.     

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.     

Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens

The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a Kd = 0.1 nM and Bmax of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC50 values of 50-120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC50 greater than or equal to 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31-36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site.     

Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins.