Biotransformation of lantadene A (22β-angeloyloxy-3-oxoolean-12-en-28-oic acid), the pentacyclic triterpenoid, by Alcaligenes faecalis

A bacterial strain capable of biotransformation oflantadene A(22-angeloyloxy-3-oxo-olean-12-en-28-oic acid),the pentacyclic hepatotoxin of lantana (Lantanacamara var. aculeata) has been isolated fromsoil using lantadene A as the sole carbon source. Theorganism is Gram negative, rod shaped, motile,catalase positive and has been identified as Alcaligenes faecalis. The isolate has been found tobe specific for lantadene A and did not utilizelantadene B. In studies using sucrose as an additionalcarbon source, A. faecalis elicitedbiotransformation of lantadene A to its trans isomer22-tigloyloxy-3-oxoolean-12-en-28-oic acid,designated as lantadene X and two other minormetabolites which could not be isolated in pure state.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Drink composition, voluntary drinking, and fluid balance in exercising, trained, heat-acclimatized boys

This study examinedthe effects of beverage composition on the voluntary drinking pattern,body fluid balance, and thermoregulation of heat-acclimatized trainedboys exercising intermittently in outdoor conditions (wet bulb globetemperature 30.4 ± 1.0°C). Twelve boys (age 13.4 ± 0.4 yr) performed two 3-h sessions, each consisting of four20-min cycling bouts at 60% maximal aerobic power alternating with25-min rest. One of two beverages was assigned: unflavored water (W) orflavored water plus 6% carbohydrate and 18 mmol/l Na (CNa). Drinkingwas ad libitum. Total intake was higher(P < 0.05) during CNa (1,943 ± 190 g) compared with W (1,470 ± 143 g). Euhydration wasmaintained with CNa (+0.18% body wt), but a mild dehydration resultedwith W (0.94% body wt; P < 0.05). Sweat loss, much higher than previously published for children of similar age, was similar between conditions (CNa = 1,644.7 ± 117.5; W = 1,750.2 ± 152.7 g). The increase in rectaltemperature (CNa = 0.86 ± 0.3; W = 0.76 ± 0.1°C), heartrate, and all perceptual variables did not differ between conditions.In conclusion, a flavored carbohydrate-electrolyte drink preventsvoluntary dehydration in trained heat-acclimatized boys exercising in atropical climate despite their large sweat losses. Because hydrationchanges were minor, the thermoregulatory strain observed was similarbetween conditions.

     

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

Generation of receptor-active, globotriaosyl ceramide/cholesterol lipid 'rafts' in vitro : A new assay to define factors affecting glycosphingolipid receptor activity

Purified renal globotriaosyl ceramide (Gb3)/cholesterol mixtures sonicated heated in a Triton-containing buffer placed below a discontinuous sucrose gradient form glycosphingolipid (GSL)-containing dense lipid structures at the 30/5% sucrose interface after centrifugation. Inclusion of fluorescein-labeled verotoxin 1 B subunit (FITC-VT1 B) within the most dense sucrose layer results in the fluorescent labeling of this Gb3-containing raft structure. Alternatively inclusion of I-labeled VT1 fractionation allows quantitation of binding. FITC-VT1 B effectively competes for I-VT1/Gb3 raft binding. This assay will allow the definition of the optimal raft composition for VT1 (or any other ligand) binding. The effect of several potential cellular raft components are reported. Increased cholesterol content increased VT1 binding. Addition of phosphatidylethanolamine had minimal effect while phosphatidylserine was inhibitory. Although inclusion of sphingomyelin increased the Gb3 content of the "raft" reduced VT1 binding was seen. Inclusion of other glycolipids can also be inhibitory. The addition of globotetraosyl ceramide had no effect; however addition of sulfogalactosyl ceramide but not sulfogalactoglycerolipid inhibited VT1/Gb3 raft binding. These results suggest that certain GSLs can disfavor the formation of the appropriate 'raft' structure for ligand binding that this is dependent on both their carbohydrate lipid structure. Such "deceptor" GSLs may provide an as yet unappreciated mechanism for the regulation of cellular GSL receptor activity. This model is an effective tool to approach the dynamics ligand-binding specificity of GSL/cholesterol-containing lipid microdomains.     

Dual role of sequence-dependent DNA curvature in nucleosome stability: the critical test of highly bent Crithidia fasciculata DNA tract

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome.     

Childhood asthma on the northern Mexico border

Children with asthma living on the northern Mexico border suffer not only from the physical aspects of this condition, but also from the lack of a clear biomedical definition and treatment plan for the illness. An ethnographic study involving participant observation and focused interviews in Tijuana, Mexico, sought to understand the intersection of diagnostic uncertainties surrounding childhood asthma on the part of parents, particularly mothers, living in acute poverty. Environmental factors such as dust and insects in impoverished homes probably acted as asthma triggers among many of the children in the study. Furthermore, management of children's asthma took place not only in biomedical clinics, but also in homes, traditional medical settings, and pharmacies, where mothers often sought remedies for their children's asthma attacks on an emergency basis. In all treatment settings, including biomedical ones, they often faced significant barriers to effective care, including the misuse of antibiotics. Thus, the role of pharmaceutical sales clerks, as well as pediatric asthma specialists, is explored in this article.     

Depressed thermogenesis but competent brown adipose tissue recruitment in mice devoid of all hormone-binding thyroid hormone receptors

We have examined the metabolic role of hormone-binding nuclear thyroid hormone receptors (TRs). Mice devoid of all hormone-binding TRs [TR alpha 1(-/-)beta(-/-) (TR-ablated mice)] had slightly decreased body temperature and much decreased basal metabolic rate, were still able to markedly increase metabolic rate in the cold, but were cold intolerant due to inadequate total heat production at low temperatures. A standard norepinephrine test showed that adrenergically induced thermogenesis could not be activated normally in the TR-ablated mice. This was not due to inadequate recruitment of brown adipose tissue, nor to the absence, decreased recruitment or dysfunction of the uncoupling protein-1. However, isolated brown fat cells were 10-fold desensitized, explaining the lack of response to standard adrenergic stimuli; cell culture experiments demonstrated that this desensitization was not an innate effect. Thus, the cold intolerance was probably not due to inadequate sympathetically induced nonshivering thermogenesis. Additionally, the results indicated that no metabolic effects of thyroid hormones could become manifest in the absence of nuclear TRs, that ligand-bound TRs were needed for euthermia and eumetabolism, but that TRs per se were not required for brown adipose tissue recruitment and uncoupling protein-1 gene expression.     

Left ventricular pressure-volume relationship in a rat model of advanced aging-associated heart failure

Aging is associated with profound changes in the structure and function of the heart. A fundamental understanding of these processes, using relevant animal models, is required for effective prevention and treatment of cardiovascular disease in the elderly. Here, we studied cardiac performance in 4- to 5-mo-old (young) and 24- to 26-mo-old (old) Fischer 344 male rats using the Millar pressure-volume (P-V) conductance catheter system. We evaluated systolic and diastolic function in vivo at different preloads, including preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (+dP/dt), and its relation to end-diastolic volume (+dP/dt-EDV) as well as the time constant of left ventricular pressure decay, as an index of relaxation. The slope of the end-diastolic P-V relation (EDPVR), an index of left ventricular stiffness, was also calculated. Aging was associated with decrease in left ventricular systolic pressure, +dP/dt, maximal slope of the diastolic pressure decrement, +dP/dt-EDV, PRSW, ejection fraction, stroke volume, cardiac and stroke work indexes, and efficiency. In contrast, total peripheral resistance, left ventricular end-diastolic volume, left ventricular end-diastolic pressure, and EDPVR were greater in aging than in young animals. Taken together, these data suggest that advanced aging is characterized by decreased systolic performance accompanied by delayed relaxation and increased diastolic stiffness of the heart in male Fischer 344 rats. P-V analysis is a sensitive method to determine cardiac function in rats.