A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum.

A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.

Heart rate as a predictor of future blood pressure in schoolchildren.

Heart rate (HR) has been shown to predict future blood pressures (BP) in studies in adults. We explored the relation of HR to future BP levels in a cohort of 344 black and 456 white schoolchildren ages 5 to 19 years, to examine the hypothesis that HR predicts subsequent BP even very early in life. After making baseline measurements, BP was assessed longitudinally 1 to 24 additional times (mean = 8.25) after the baseline period, at intervals of approximately 6 months. We found that HR was significantly related to future diastolic BP in the black boys (P = .016) after adjusting for baseline diastolic BP, age, and body mass index, but not in the black girls or in the white children. Because HR is reflective of sympathetic nervous system (SNS) activity that in turn can be related to the renin-angiotensin system (RAS), we also explored the relation of HR to the RAS by studying relationships to variants in the angiotensinogen gene and the angiotensin I-converting enzyme (ACE) gene. We found a significantly positive relationship of HR to the presence of the deletion allele of the ACE gene (P = .0015), but, again, only in the black boys. Because blacks in general appear to retain additional sodium when compared with whites, the SNS, as reflected in the HR, may influence BP more when individuals have increased sodium retention. In summary, baseline HR predicted future diastolic BP in the black boys but not in the black girls or in the white children.

Molecular variations in the calcium-sensing receptor in relation to sodium balance and presence of hypertension in blacks and whites.

Sodium (Na) excretion is to an extent tied to calcium (Ca) excretion; increases in Ca result in increased Na excretion. We hypothesized that molecular variation in the calcium-sensing receptor (CaSR), which imparts certain of the influences of extracellular Ca, might be related to differences in Na balance and blood pressure. We further hypothesized that such an influence by CaSR is more pronounced in blacks than in whites, as the hypertension in blacks appears to be more dependent on Na retention. Three common molecular variants in CaSR were studied. Two were more frequent in the whites (A986S, P < .0001, and G990R, P = .093), whereas Q1011E was more frequent in the blacks (P < .0001). Two distinctly separate groups were studied: (1) healthy schoolchildren in whom levels of the renin-aldosterone axis and blood pressure were measured, and (2) normotensive and hypertensive adults. Studies of association were made separately in the whites and the blacks. No association of any of the variants with Na balance (as estimated from renin and aldosterone levels) was observed. In the black schoolchildren, Q1011E showed a marginal association with a higher blood pressure (P = .093 for systolic and P = .025 for diastolic), a relationship that was considered to be nonsignificant after adjusting for multiple comparisons. Nor was there a significant association of the variants with presence or absence of hypertension. In summary, studies of two cohorts that included whites and blacks did not suggest that molecular variations in the CaSR influence either Na balance or blood pressure.

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants.

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O >> NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.

Monomeric sarcosine oxidase: 2. Kinetic studies with sarcosine, alternate substrates, and a substrate analogue.

Monomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. MSOX can oxidize other secondary amino acids (N-methyl-L-alanine, N-ethylglycine, and L-proline), but N,N-dimethylglycine, a tertiary amine, is not a substrate. N-Methyl-L-alanine is a good alternate substrate, exhibiting a k(cat) value (8700 min(-)(1)) similar to sarcosine (7030 min(-)(1)). Turnover with L-proline (k(cat) = 25 min(-)(1)) at 25 degrees C occurs at less than 1% of the rate observed with sarcosine. MSOX is converted to a two-electron reduced form upon anaerobic reduction with sarcosine or L-proline. No evidence for a spectrally detectable intermediate was obtained in reductive half-reaction studies with L-proline. The reductive half-reaction with L-proline at 4 degrees C exhibited saturation kinetics (k(lim) = 6.0 min(-)(1), K(d) = 260 mM) and other features consistent with a mechanism in which a practically irreversible reduction step (E(ox). S --> E(red).P) with a rate constant, k(lim), is preceded by a rapidly attained equilibrium (K(d)) between free E and the E.S complex. Steady-state kinetic studies with sarcosine and N-methyl-L-alanine in the absence or presence of a dead-end inhibitor (pyrrole-2-carboxylate) indicate that catalysis proceeds via a "modified" ping pong mechanism in which oxygen reacts with E(red).P prior to the dissociation of the imino acid product. In this mechanism, double reciprocal plots will appear nearly parallel (as observed) if the reduction step is nearly irreversible. A polar mechanism, involving formation of a covalent 4a-flavin-substrate adduct is one of several plausible mechanisms for sarcosine oxidation. Thiols are known to form similar 4a-flavin adducts. MSOX does not form a 4a-adduct with thioglycolate but does form a charge-transfer complex that undergoes an unanticipated one-electron-transfer reaction to yield the anionic flavin radical.

Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension.

Renin and aldosterone secretion is often lower in blacks than in whites, characteristics that resemble a milder form of Liddle syndrome in which a mutation in the amiloride-sensitive epithelial sodium channel (ENaC) of the kidney results in enhanced resorption of sodium. In the present study, we looked for evidence that the intrinsic level of ENaC activity is indeed higher in blacks than in whites. In overnight urine samples collected from young people (249 white and 181 black subjects, mean age 13.4 years), the urinary aldosterone/potassium ratio, which is typically very low in Liddle syndrome, was lower in blacks than in whites: 0.421+/-0.024 (mean+/-SE) versus 0.582+/-0.016 nmol/mmol (P<0.0001). In addition, all but 1 of 5 molecular variants in ENaC were much more common in blacks than in whites. G442V in the beta-subunit, present in 16% of the blacks and in only 1 white, was associated with parameters reflective of a greater Na retention and potentially a higher ENaC activity: a lower plasma aldosterone concentration (P=0.070), a lower urinary aldosterone excretion rate (P=0.052), a higher potassium excretion rate (P=0.048), and a lower urinary aldosterone/potassium ratio (P=0.027). In a second cohort consisting of 126 black and 161 white normotensive subjects and 232 black and 188 white hypertensive subjects, betaG442V did not show a significant association with hypertension (P=0.089). On the other hand, a variant that was twice as common in whites, alphaT663A, was associated with being normotensive both in blacks (P=0.018) and in whites (P=0.034). Expression of either betaG442V or alphaT663A in Xenopus oocytes did not result in a change in basal Na current, consistent with the variants being in linkage disequilibrium with alleles at active loci. In conclusion, several lines of evidence are presented to suggest that ENaC activity is higher in blacks than in whites, which could contribute to racial differences in Na retention and the risk for hypertension.

Potential involvement of calcium, CaM kinase II, and MAP kinases in PCB-stimulated insulin release from RINm5F cells.

Polychlorinated biphenyls (PCBs) are environmental contaminants that induce release of insulin in rat insulinoma cells, RINm5F (Fischer et al., Life Sci. (1996) 59, 2041-2049). In the present study the mechanisms of this effect were investigated using noncytotoxic concentrations (10 microg/ml) of a PCB mixture, Aroclor-1254, and the pure PCB congeners 2,2',4,4'-tetrachlorobiphenyl and 2,2',4,4',5, 5'-hexachlorobiphenyl. Treatment of RINm5F cells with each of these agents resulted in a rapid increase in intracellular free calcium. The presence of extracellular calcium was required for PCB-induced insulin release because removal of calcium from the medium attenuated the effect. In addition, pretreatment of RINm5F cells with the calcium channel blocker verapamil also blocked PCB-induced insulin release. To determine whether PCB-related insulin release could be associated with the enzyme, calcium/calmodulin-dependent kinase II (CaM kinase II), RINm5F cells were pretreated with the CaM kinase II inhibitor KN-93. PCB-induced insulin release was completely blocked by KN-93. Under similar treatment conditions, PCBs also induced the activity of mitogen-activated protein kinases (MAPK) 1 and 2. However, inhibition of MAPK activation by a specific inhibitor, PD-98059 (10.0 microM) did not prevent insulin release induced by PCBs. The results of the present investigation suggest a role for calcium and CaM kinase II in PCB-induced insulin release. Furthermore, the results suggest that insulin release by PCBs is independent of the activation of MAPKs.

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase.

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.

Serum angiotensinogen concentration in relation to gonadal hormones, body size, and genotype in growing young people.

Multiple factors are thought to influence the level of circulating angiotensinogen (AGT). We showed previously that the serum AGT concentration was significantly related to body mass index (BMI) in a cohort of young people. In the present study, we studied whether levels of the gonadal hormones estradiol and testosterone might also predict the AGT level and might contribute to the BMI effect, since both the production of these hormones and BMI increase with age. In boys (n=127; mean+/-SD age, 14.7+/-1.9 years) and girls (n=104; age, 14.8+/-1.9 years) studied as a single group, we found a significant association of AGT level with level of estradiol (P=0.015) after adjustment for haplotype, age, race, testosterone concentration, and BMI. In girls studied alone, the level of AGT showed a significantly positive relation to level of testosterone (P=0.043), possibly a result of peripheral conversion of testosterone to estradiol, after adjustment for haplotype, age, race, estradiol concentration, and BMI. In boys, on the other hand, the level of testosterone was inversely related to AGT concentration (P=0.019), again after making adjustments for the other variables. Finally, in pairs of subjects matched for BMI, age, race, and gender where 1 member of each pair had either 1 or 2 copies of an AGT gene haplotype (T235 and -1074t) and the other member had no copy, the level of AGT was higher in the carrier of a haplotype in 24 of the 34 pairs (P<0.001). In conclusion, gonadal hormones are an additional influence on the circulating level of AGT in growing young people. In addition, with matching for BMI and other covariates, there is a strong association of AGT genotype with the serum level of AGT, emphasizing the importance of AGT gene expression as a determinant of the circulating level of AGT.

Folate utilization by monomeric versus heterotetrameric sarcosine oxidases.

There are two types of bacterial sarcosine oxidases. The heterotetrameric enzymes contain subunits ranging in size from about 10 to 100 kDa, noncovalently bound FAD and NAD+, and covalently bound FMN attached to the beta subunit (42-45 kDa). Monomeric sarcosine oxidases are similar in size to the beta subunit in the heterotetramers and contain covalently bound FAD. Formaldehyde formation during sarcosine oxidation by several heterotetrameric sarcosine oxidases was suppressed in the presence of 50 microM [6S]-tetrahydrofolate, accompanied by a 25-50% increase in the rate of sarcosine oxidation. In contrast, [6S]-tetrahydrofolate caused only a modest decrease in the rate of formaldehyde production with monomeric sarcosine oxidases (approximately 25%), an effect which was virtually entirely attributable to an accompanying decrease in the rate of sarcosine oxidation. In the presence of 100 microM [6R,S]-tetrahydropteroyltriglutamate [H4Pte(Glu)3], the heterotetrameric enzymes catalyzed the formation of 5,10-methylenetetrahydropteroyltriglutamate [5,10-CH2-H4Pte(Glu)3] at a rate which was 35-60% faster than the rate of sarcosine oxidation in the absence of folate. An apparent Km value of 3.1 microM was estimated for [6S]-H4Pte(Glu)3 with the heterotetrameric corynebacterial sarcosine oxidase. In contrast, slow formation of 5,10-CH2-H4Pte(glu)3 was detected during sarcosine oxidation with monomeric sarcosine oxidases, attributable to the nonenzymatic reaction of free formaldehyde with H4Pte(Glu)3. The results show that only the heterotetrameric sarcosine oxidases can use tetrahydrofolates as substrates and, in this regard, they resemble mammalian sarcosine and dimethylglycine dehydrogenases.

Association of retinol binding protein in multiple-case families with insulin-dependent diabetes.

We performed a family study to investigate the heritability of reduced serum retinol levels observed in type 1 diabetes cases. Diet and serum factors, including retinol, total carotene, malondialdehyde, and retinol binding protein levels, were measured in 11 multiple-case families. The mean serum retinol level of the diabetics (46 ug/dl) was significantly less than the mean serum retinol level of the nondiabetics (60.9 ug/dl). The level of retinol binding protein was also significantly lower in diabetics (6.2 mg/dl) than in nondiabetics (7.6 mg/dl). The serum values of retinol binding protein were closely related within families, including both diabetic and nondiabetic family members. A characteristic shared between diabetics and one-third of their family members was a low ratio of serum retinol to total carotene, suggesting a low conversion of dietary carotene into retinol. Analysis of food frequency reports showed no difference between dietary retinol or total carotene level in diabetics or their relatives. This study offers evidence that heritability and the reduced conversion of carotene may play a role in the level of serum retinol in type 1 diabetes cases.

Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic acid synthesis.

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus--all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.

Polychlorinated biphenyls release insulin from RINm5F cells.

Polychlorinated biphenyls (PCBs) possess a variety of biological effects, including alterations in growth, development and metabolism, that may be dependent on insulin. However, no reports on the action of PCBs on cells which produce and secrete insulin are available. The current study examined the ability of a commercial mixture of PCBs (Aroclor 1254) and three specific PCB congeners, to alter the release of insulin using the hormone producing cell line RINm5F. Exposure of cells to Aroclor 1254 (A-1254) produced a concentration-dependent increase in media insulin reaching a peak, when expressed as percent of control, at 30 min. In spite of continued exposure, media insulin relative to control declined and no treatment-related difference was observed at 48 hrs. Cellular levels of the hormone declined as much as 50% by that time. The insulin releasing action of A-1254 was mimicked by each of the non-coplanar congeners 2,2',4,4'-tetrachlorobiphenyl (TCB) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) but the coplanar congener 3,3',4,4'-TCB showed no significant activity. These results indicate that PCBs are capable of producing a release of insulin from RINm5F cells, an effect that is unlikely to be associated with coplanar congeners that initiate their action by binding to the Ah-receptor.

Possible roles for glucocorticoids and apoptosis in the suppression of lymphopoiesis during zinc deficiency: a review.

Thymic atrophy and lymphopenia are immunological hallmarks of many forms of malnutrition including deficiencies in zinc. Extreme thymic atrophy (70-80%) along with a 50% loss of splenocytes in mice maintained on a zinc deficient diet (ZD) for 30 days suggested that the deficiency might be altering lymphopoiesis or the production of new lymphocytes by the bone marrow. As shown herein, mice who were marginally zinc deficient being 72-75% the body weight of adequately fed controls, exhibited a 50% decline in pre B-cells and a 25% decline in immature B-cells. The mature B-cells of the marrow appeared fairly resistant to effects of suboptimal zinc intake. Interesting, this pattern was similar to results obtained by treating bone marrow cells with levels of glucocorticoids analogous to those found in nutritionally deficient rodents. Furthermore, these same concentrations of steroids were shown to induce significant levels of apoptosis or cell death among pre and immature B-cells which accounted for their declining numbers subsequent to exposure to glucocorticoid. In order to better ascertain the potential role of glucocorticoids generated during zinc deficiency on lymphopoietic processes, adrenalectomies were performed in an attempt to remove glucocorticoids from the equation. Subsequently, adrenalectomized and sham operated mice were placed on a ZD or zinc adequate diet (ZA). Levels of steroids at the time of sacrifice were elevated six fold in non-adrenalectomized ZD mice compared to ZD adrenalectomized mice. Removal of the adrenal gland protected the thymus of ZD mice from atrophy and also provided substantial protection of lymphopoietic processes.(ABSTRACT TRUNCATED AT 250 WORDS)

The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture.

The mammalian organ of Corti has one of the most highly ordered patterns of cells in any vertebrate sensory epithelium. A single row of inner hair cells and three or four rows of outer hair cells extend along its length. The factors that regulate the formation of this strict pattern are unknown. In order to determine whether retinoic acid plays a role during the development of the organ of Corti, exogenous retinoic acid was added to embryonic mouse cochleae in vitro. Exogenous retinoic acid significantly increased the number of cells that developed as hair cells and resulted in large regions of supernumerary hair cells and supporting cells containing two rows of inner hair cells and up to 11 rows of outer hair cells. The effects of retinoic acid were dependent on concentration and on the timing of its addition. Western blot analysis indicated that cellular retinoic acid binding protein (CRABP) was present in the sensory epithelium of the embryonic cochlea. The amount of CRABP apparently increased between embryonic day 14 and postnatal day 1, but CRABP was not detectable in sensory epithelia from adults. A retinoic acid reporter cell line was used to demonstrate that retinoic acid was also present in the developing organ of Corti between embryonic day 14 and postnatal day 1, and was also present in adult cochleae at least in the vicinity of the modiolus. These results suggest that retinoic acid is involved in the normal development of the organ of Corti and that the effect of retinoic acid may be to induce a population of prosensory cells to become competent to differentiate as hair cells and supporting cells.

Asymmetrical retinoic acid synthesis in the dorsoventral axis of the retina.

An aldehyde dehydrogenase present at high levels in the dorsal retina of the embryonic and adult mouse was identified as the isoform AHD-2 known to oxidize retinaldehyde to retinoic acid. Comparative estimates of retinoic acid levels with a reporter cell line placed the retinas among the richest tissues in the entire body of the early embryo; levels in ventral retina, however, exceeded dorsal levels. Retinoic acid synthesis from retinaldehyde in the dorsal pathway was less effective than the ventral pathway at low substrate levels and more effective at high levels. The dorsal pathway was preferentially inhibited by disulfiram, while ventral synthesis was preferentially inhibited by p-hydroxymercuribenzoate. When protein fractions separated by isoelectric focusing were analyzed for retinoic acid synthesizing capacity by a zymography-bioassay, most of the synthesis in dorsal retina was found to be mediated by AHD-2, and ventral synthesis was mediated by dehydrogenase activities distinct in charge from AHD-2. Postnatally, levels of highest retinoic acid synthesis shifted from ventral to dorsal retina. In the adult retina, the dorsal pathway persisted, but the preferential ventral pathway was no longer detectable. Our observations raise the possibility that retinoic acid plays a role in the determination and maintenance of the dorsoventral axis of the retina, and that the morphogenetically significant asymmetry here lies in the spatial arrangement of synthetic pathways.

Adrenal androgen excretion during adrenarche. Relation to race and blood pressure.

We have previously shown that black children have higher blood pressures than white children. In the present study, we examined whether a possible racial difference in adrenal androgen production during adrenarche might contribute to the racial disparity in blood pressure. Adrenal androgen production was estimated from urinary excretion of adrenal androgen metabolites that showed cross-reactivity with antisera to dehydroepiandrosterone sulfate (DHEA-S). Urine samples were collected overnight in 798 children, one third of whom were black. Analyses were performed for two different age groups, less than 10 years and 10 years or more of age. In children less than 10 years of age, adrenal androgen excretion rates were 17% higher in blacks than in whites (p = 0.0099); adrenal androgen excretion rates tended to be higher in older black children as well, but differences here were not statistically significant. Adrenal androgen excretion rates were positively correlated with diastolic blood pressure in the older age group only (p = 0.014). However, when the relation of race to blood pressure was examined along with adrenal androgen excretion adjusted for age, sex, and weight, race remained an independent contributor to the level of blood pressure, suggesting that a difference in adrenal androgens could not explain the racial differences in blood pressure. In summary, black children produced more adrenal androgen, but this did not explain their higher blood pressures. In older children, where adrenal androgen excretion rates were higher, diastolic blood pressure and adrenal androgen excretion were positively related, suggesting that adrenal androgens participate in establishing the level of blood pressure in young people.

Enhanced susceptibility to sepsis after simple hemorrhage. Depression of Fc and C3b receptor-mediated phagocytosis.

To determine whether phagocytosis mediated by Fc receptors and/or receptors for the third component of complement (C3b) are altered after hemorrhage, C3H/HeN mice were subjected to nonlethal hemorrhage and then adequately resuscitated. Twelve hours after the hemorrhagic episode, a significant decrease in both Fc (-55.2%) and C3b (-46.6%) receptor-positive peritoneal macrophages was observed compared with controls. At 24 hours the extent of the depression, while still marked, was only -22.5% and -17.4% for Fc and C3b receptors, respectively. By day 3 after hemorrhage, no differences could be observed for either of these receptors. The capacity of macrophages from mice after hemorrhage to elaborate interleukin 1 or tumor necrosis factor-alpha showed no increase over that of the sham controls, and serum levels of endotoxin were not elevated 2 or 24 hours after hemorrhage. Moreover, endotoxin-tolerant C3H/HeJ mice also exhibited depression of both receptors after hemorrhage. Thus, the inability of the host macrophages to clear opsonized infectious agents after hemorrhage may be due in part to the loss of Fc and C3b receptors on macrophages.