Xenobiotic oxidation by cytochrome P-450-enriched extracts of Streptomyces griseus

Crude extracts of Streptomyces griseus grown on soybean flour-enriched medium contain high levels of cytochrome P-450. The cytochrome P-450-enriched fractions, obtained by ammonium sulfate fractionation (30-50% saturation), catalyze the NADPH-dependent oxidation of a variety of xenobiotics when complemented with both spinach ferredoxin:NADP+ oxidoreductase and spinach ferredoxin. Reactions observed are aromatic, benzylic and alicyclic hydroxylations, O-dealkylation, non-aromatic double bond epoxidation, N-oxidation and N-acetylation.     

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: Construction of models and comparison with experiment

Homology modeling methods have been used to construct models of two proteins—the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions—possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled—reasons for this and possible enhancements are discussed. © 1995 Wiley-Liss, Inc.     

Further studies of the action of disulfiram and 2,2''-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase.

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2'-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2'-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.     

Synthesis of methyl 2- and 4-pyridyl disulfide from 2- and 4-thiopyridone and methyl methanethiosulfonate

In 1982, methyl 2-pyridyl disulfide was reported as a new reagent for the titration of thiol groups in peptides and proteins and for their temporary blocking with the thiomethyl group [T. Kimura et al. (1982) Anal. Biochem. 122, 274-282]. We have synthesized this compound (and its 4-pyridyl isomer) by a rapid and convenient procedure which is preferable to that in the original report. Our method involves the thiomethylation of the appropriate thiopyridone by methyl methanethiosulfonate.     

Utilization of milk energy by suckling mink kits

A total of 36 mink dams and their litters of 3, 6 or 9 kits were used for determination of milk intake of the suckling young by means of deuterium dilution technique, and chemical composition of milk and of kit bodies. Measurements were performed during lactation weeks 1?–?4, each week with 3 dams with each litter size. Milk intake was determined over a 48?h measurement period, and by the end of this milk samples were collected and 2 kits (litters of 6 and 9) or 1 kit per litter (litters of 3) were killed for body chemical composition. Based on the results, different models were applied for calculation of the energetic efficiency of milk. Dam milk yield increased steadily from week 1 until week 3 but only slightly from week 3 to 4. The increase declined with increasing litter size, and for dams suckling 9 kits the increment from week 3 to week 4 was only 2?g. The dry matter content of milk increased significantly as lactation progressed, being reflected in crude protein increasing from 6.9% in lactation week 1 to 8.1% in week 4. Milk fat increased concomitantly from 5.6% to 8.0%. In kit bodies, crude protein content increased from 9.4% in week 1 to about 12% in weeks 3 and 4. Body fat content increased from week 1 (4.1%) to week 3 (8.4%) and then declined in week 4 (7.1%). Animals suckled in litters of 3 kits had the highest milk intake and live weight and kits suckled in litters of 9 had the lowest milk intake, live weight and daily gain. In terms of milk intake per g gain kits in litters of 6 were the most efficient, with 4.1?g milk per g body gain. The metabolizable energy requirement for maintenance (MEm) was estimated to 448 kJ/kg^0.75 and the efficiency of utilization of ME for body gain (k_g) to 0.67, the estimates being higher (MEm) or in good agreement with previous findings (k_g) in suckling mink kits.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074     

Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL. Subsequent studies revealed a reduction of the mitochondrial membrane potential and caspase-3 activation in these two cell lines upon treatment with proteasome inhibitors; however, caspase-3 activation occurred much earlier in Jurkat cells. Cell cycle analysis indicated a sub-G(1) apoptotic cell population in Jurkat cells and G(2)/M arrest in YT cells after they were treated by proteasome inhibitors. Moreover, pretreatment of YT cells by a caspase inhibitor followed by a proteasome inhibitor did not increase the percentage of G(2)/M phase cells. In addition, accumulation of p27 and IkappaB-alpha was detected only in Jurkat cells, but not YT cells. In summary, proteasome inhibitors may act differentially in cell cycle arrest and apoptosis of tumors of NK and T cell origin, and may have similar effects on normal NK and T cells.     

Fluctuating asymmetry in bank vole populations (Rodentia, Arvicolinae) reflects stress caused by landscape fragmentation in the Mont-Saint-Michel Bay

In intensively farmed, reclaimed areas (polders) of Mont-St-Michel Bay, France, bank voles ( Clethrionomys glareolus ) live in fragmented hedgerows, where populations are small and dispersal rates and genetic diversity are low. These small populations are likely to have been exposed to potential environmental and/or genetic stress. The sensitivity of development to stress can be measured by fluctuating asymmetry (FA). FA was calculated for three samples from a disturbed area and one sample from an adjacent, more connected and undisturbed landscape. Size FA was estimated from 16 measurements of the skull and teeth whilst shape asymmetry was estimated from the skull alone. Bank voles in fragmented hedgerows of the disturbed area had a higher degree of FA than bank voles from the more extensive and more connected hedges of the undisturbed area. These results were confirmed by the study of shape asymmetry, body mass and centroid size of the skull. There were no differences in FA between the three disturbed area samples. We conclude that FA does not reveal differences in the development of bank voles living in isolation under different local conditions in the various parts of the disturbed area. However, FA may allow differentiation between populations from greatly contrasting landscapes.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80, 37–44.