Purification and characterization of aldo-keto reductases from gerbil liver: immunochemical evidence for related proteins in other mammalian species

We purified a hepatic aldehyde reductase (AR1) and two carbonyl reductases (CR1, CR2) from the Mongolian gerbil, an animal recently shown to closely resemble man in its metabolism of a carbonyl containing organochlorine pesticide. The apparent molecular weights of AR1, CR1, and CR2 were 40,700, 33,000, and 34,700, respectively. Typical of similar enzymes in other species, gerbil AR1 reduced aliphatic and aromatic aldehydes and was inhibited by phenobarbital or valproate, whereas CR1 and CR2 catalyzed the reduction of aromatic aldehydes and ketones as well as quinones and were inhibited by p-chloromercuribenzoate, mercuric chloride, or pyrazole. All three enzymes were insensitive to metal chelating agents and utilized NADPH as their cofactor. CR1 was unique in being equally active with NADH as its cofactor. Antibodies raised against CR1 reacted with purified CR1 and CR2, but not with AR1, as judged by immunoblot analyses. There were three immunochemically related proteins in gerbil liver cytosol (30 to 35 kDa range) recognized by the anti-CR1 IgG. Similar immunoblot analyses of hepatic cytosolic proteins from other mammalian species revealed immunoreactive proteins only in the hamster, the rabbit, and man, and not in the rat, the mouse, or the guinea pig. Quantitative immunoblot analyses of human liver cytosol from seven patients revealed three immunoreactive proteins. These were present in unequal and varying concentrations, although there were only small interindividual differences in the total concentration of the immunoreactive proteins. We conclude that there are multiple molecular forms of immunochemically related hepatic carbonyl reductases in the gerbil and in some other mammalian species, including man.     

酿酒酵母孢子壁结构及其合成相关基因研究进展

细胞壁是酵母细胞区别于哺乳动物细胞的重要特征结构。酵母细胞壁的结构组成、合成、再生等与酵母自身繁殖及环境胁迫压力密切相关。目前,酵母孢子壁的形成机理、调控过程机制及孢子壁合成相关基因的功能尚未研究清楚。本文以酿酒酵母为例,简要描述酵母孢子壁的形成过程,重点阐述孢子壁甘露糖层、葡聚糖层、壳聚糖层和二酪氨酸层的结构组成及其合成相关基因的国内外研究进展,以期为抗真菌药物的新靶点研究提供参考。     

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.     

Rationale and design of EXPLORE: a randomized, prospective, multicenter trial investigating the impact of recanalization of a chronic total occlusion on left ventricular function in patients after primary percutaneous coronary intervention for acute ST-elevation myocardial infarction

Background

Hypothyroidism is a prevalent endocrine condition. Individuals with this disease are commonly managed through supplementation with synthetic thyroid hormone, with the aim of alleviating symptoms and restoring normal thyroid stimulating hormone levels. Generally this management strategy is effective and well tolerated. However, there is research to suggest that a significant proportion of hypothyroid sufferers are being inadequately managed. Furthermore, hypothyroid patients are more likely to have a decreased sense of well-being and more commonly experience constitutional and neuropsychiatric complaints, even with pharmacological intervention. The current management of hypothyroidism follows a biomedical model. Little consideration has been given to a biopsychosocial approach to this condition. Within the chiropractic profession there is growing support for the use of a biopsychosocial-based intervention called Neuro-Emotional Technique (NET) for this population.

Methods/Design

A placebo-controlled, single-blinded, randomised clinical pilot-trial has been designed to assess the influence of Neuro-Emotional Technique on a population with primary overt hypothyroidism. A sample of 102 adults (≥18 years) who meet the inclusion criteria will be randomised to either a treatment group or a placebo group. Each group will receive ten treatments (NET or placebo) over a six week period, and will be monitored for six months. The primary outcome will involve the measurement of depression using the Depression, Anxiety and Stress Scale (DASS). The secondary outcome measures to be used are; serum thyroid stimulating hormone, serum free-thyroxine, serum free-triiodothyronine, serum thyroid peroxidase auto-antibodies, serum thyroglobulin auto-antibodies as well as the measurement of functional health and well-being using the Short-Form-36 Version 2. The emotional states of anxiety and stress will be measured using the DASS. Self-measurement of basal heart rate and basal temperature will also be included among the secondary outcome measures. The primary and secondary measures will be obtained at commencement, six weeks and six months. Measures of basal heart rate and basal temperature will be obtained daily for the six month trial period, with recording to commence one week prior to the intervention.

Discussion

The study will provide information on the influence of NET when added to existing management regimens in individuals with primary overt hypothyroidism.

Trial Registration

ANZCTR Number: 12607000040460     

Characterization of chaotic dynamics in the human menstrual cycle

Background  

The human menstrual cycle is known to exhibit a significant amount of unexplained variability. This variation is typically dismissed as random fluctuations in an otherwise periodic and predictable system. Given the many delayed nonlinear feedbacks in the multiple levels of the reproductive endocrine system, however, the menstrual cycle can properly be construed as the output of a nonlinear dynamical system, and such a system has the possibility of being in a chaotic trajectory. We hypothesize that this is in fact the case and that it accounts for the observed variability.     

Optimizing harvest of corn stover fractions based on overall sugar yields following ammonia fiber expansion pretreatment and enzymatic hydrolysis

Background  

Corn stover composition changes considerably throughout the growing season and also varies between the various fractions of the plant. These differences can impact optimal pretreatment conditions, enzymatic digestibility and maximum achievable sugar yields in the process of converting lignocellulosics to ethanol. The goal of this project was to determine which combination of corn stover fractions provides the most benefit to the biorefinery in terms of sugar yields and to determine the preferential order in which fractions should be harvested. Ammonia fiber expansion (AFEX) pretreatment, followed by enzymatic hydrolysis, was performed on early and late harvest corn stover fractions (stem, leaf, husk and cob). Sugar yields were used to optimize scenarios for the selective harvest of corn stover assuming 70% or 30% collection of the total available stover.     

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice

Background

The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood.

Results

We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region.

Conclusion

We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.     

Purification and characterization of chlordecone reductase from human liver

The toxic organochlorine pesticide, chlordecone (Kepone), is excreted in human bile primarily as a stable, reduced monoalcohol metabolite. This bioreduction is catalyzed by a hepatic cytosolic enzyme activity termed chlordecone reductase. We purified this enzyme from human liver and found that chlordecone reductase resembles the family of xenobiotic metabolizing enzymes referred to as the aldo-keto reductases based on its biochemical characteristics, including its ability to catalyze the reduction of a carbonyl-containing substrate. However, analyses of liver cytosolic samples on immunoblots developed with anti-chlordecone reductase antibodies revealed that immunoreactive proteins were present only in those mammalian species that convert chlordecone to chlordecone alcohol in vivo (man, gerbil, and rabbit) and not in those species unable to reduce chlordecone (rat, mouse, and hamster). Hence, chlordecone reductase is unique among aldo-keto reductases in being species-specific. Quantitative immunoblot analyses of seven human liver specimens disclosed two immunoreactive proteins whose total concentration varied over a 6-fold range. Moreover, the amount of immunoreactive protein was directly proportional to chlordecone reductase activity in each sample. We conclude that chlordecone reductase is a unique aldo-keto reductase of potential clinical importance whose expression varies markedly among individuals.     

Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture

Treatment of rats with macrolide antibiotics such as triacetyloleandomycin (TAO) dramatically increases the hepatic concentration of a cytochrome P-450 indistinguishable from P-450p, the major liver cytochrome induced by glucocorticoids such as dexamethasone (Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., Young, B., and Guzelian, P. S. (1985) Biochemistry 24, 2171-2178). To investigate the mechanism of induction of P-450p, we treated rats for 4 days with these agents and found that dexamethasone and TAO induced the synthesis of P-450p at least 70- and 35-fold over control values, respectively, as estimated from measurements of P-450p mRNA translatable in a cell-free system. However, the accumulation of P-450p holocytochrome (measured as TAO-metabolite spectral complex) or P-450p protein (measured by quantitative immunoblotting) increased at least 150-fold by TAO but only 32-fold by dexamethasone. The possibility that TAO decreases the degradation of P-450p was supported by the observation that administration of TAO to dexamethasone-treated rats labeled with NaH[14C]O3 and [3H]-delta-aminolevulinic acid retarded the decay of radioactive immunoprecipitable P-450p protein (t1/2 = 60 versus 14 h) and heme (t1/2 = 73 versus 10 h). To confirm these results, P-450p protein synthesis was measured as radioactivity incorporated into immunoprecipitable P-450p in primary monolayer cultures of adult rat hepatocytes incubated with [3H]leucine. Dexamethasone treatment of the cultures stimulated P-450p synthesis by at least 30-fold whereas macrolides were without effect. However, macrolide antibiotics but not dexamethasone inhibited the disappearance of radiolabeled P-450p from cultured hepatocytes similar to the results obtained in vivo. We conclude that macrolide antibiotics induce P-450p, the most rapidly turning over cytochrome yet reported, by stimulating its synthesis indirectly and by blocking its degradation, significantly.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.