Foetal exposure to phthalate esters and anogenital distance in male newborns.

Phthalate esters, commonly used as plasticizers, show anti-androgenic activity and cause male reproductive malformation in experimental animals. However, the effects of prenatal exposure to phthalate esters in humans have not been extensively studied. The purpose of this study was to examine the relationship between prenatal exposure to phthalate esters and the anogenital distance (AGD) as a reproductive endpoint in human male newborns. Spot urine samples were collected from 111 Japanese pregnant women after obtaining their informed consent. Seven urinary phthalate ester metabolites were determined by high performance liquid chromatography-tandem mass spectrometry. Urinary isoflavones concentrations were measured as possible covariates because their oestrogenicities and high exposure levels among Japanese have the potential to affect male genital development. Birth outcomes and AGD, the distance from the centre of the anus to external genitalia, were measured for their male newborns. In a multiple regression model, the log-transformed mono-2-ethylhexyl phthalate concentration (specific gravity-corrected) was negatively significant, and maternal smoking status was positively significant, in explaining anogenital index (AGI) when potential covariates were controlled for. Urinary isoflavones did not significantly contribute to AGI in any models. Our results suggest that prenatal exposure to di(2-ethylhexyl) phthalate affects reproductive development in human males.

Beta amyloid deposition and neurofibrillary tangles spontaneously occur in the brains of captive cheetahs (Acinonyx jubatus).

Alzheimer disease is a dementing disorder characterized pathologically by Aß deposition, neurofibrillary tangles, and neuronal loss. Although aged animals of many species spontaneously develop Aß deposits, only 2 species (chimpanzee and wolverine) have been reported to develop Aß deposits and neurofibrillary tangles in the same individual. Here, the authors demonstrate the spontaneous occurrence of Aß deposits and neurofibrillary tangles in captive cheetahs (Acinonyx jubatus). Among 22 cheetahs examined in this study, Aß deposits were observed in 13. Immunostaining (AT8) revealed abnormal intracellular tau immunoreactivity in 10 of the cheetahs with Aß deposits, and they were mainly distributed in the parahippocampal cortex and CA1 in a fashion similar to that in human patients with Alzheimer disease. Ultrastructurally, bundles of straight filaments filled the neuronal somata and axons, consistent with tangles. Interestingly, 2 of the cheetahs with the most severe abnormal tau immunoreactivity showed clinical cognitive dysfunction. The authors conclude that cheetahs spontaneously develop age-related neurodegenerative disease with pathologic changes similar to Alzheimer disease.

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb.

We have characterized two separate odorant receptor (OR) gene clusters to examine how olfactory neurons expressing closely linked and homologous OR genes project their axons to the olfactory bulb. Murine OR genes, MOR28, MOR10, and MOR83, share 75-95% similarities in the amino acid sequences and are tightly linked on chromosome 14. In situ hybridization has demonstrated that the three genes are expressed in the same zone, at the most dorsolateral and ventromedial portions of the olfactory epithelium, and are rarely expressed simultaneously in individual neurons. Furthermore, we have found that olfactory neurons expressing MOR28, MOR10, or MOR83 project their axons to very close but distinct subsets of glomeruli on the medial and lateral sides of the olfactory bulb. Similar results have been obtained with another murine OR gene cluster for A16 and MOR18 on chromosome 2, sharing 91% similarity in the amino acid sequences. These results may indicate an intriguing possibility that olfactory neurons expressing homologous OR genes within a cluster tend to converge their axons to proximal but distinct subsets of glomeruli. These lines of study will shed light on the molecular basis of topographical projection of olfactory neurons to the olfactory bulb.

Simplified method of determination of serum cholesterol sulfate by reverse phase thin-layer chromatography.

A new method for determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) from 1 ml serum by reverse phase thin-layer chromatography (TLC) is described. The method comprises an isolation step of sulfated steroids by means of octadecylsilane-bonded (C18) reverse phase column chromatography, a solvolysis step for desulfation of sulfated steroids, and a C18 TLC step for measurement on a photodensitometer. This method is much simpler and more rapid than the methods previously reported, since neither a radioisotope is needed, nor any steps of saponification, derivatization, tedious scraping from a TLC plate, and time-consuming conventional column chromatography are not required. The present method allowed us to distinguish recessive X-linked ichthyosis (RXLI) very easily from ichthyosis vulgaris (IV) by the size and gradation of clearly visible blue chromogen derived from CS on a TLC plate in RXLI. By photodensitometer scanning, the CS levels in patients with RXLI were about 10 times higher than those of patients with IV and healthy subjects, whereas the DHEAS level was normal in the RXLI patients. The present simplified method proved to be useful in diagnosis of RXLI.

Effect of aging on sebaceous gland activity and on the fatty acid composition of wax esters.

Using fused-silica capillary gas chromatography, we investigated sebum samples from 55 healthy individuals to discover the effects of aging on the sebaceous gland activity and on the fatty acid composition of wax exters. The sebaceous gland activity, which was expressed by the ratio of wax esters/[cholesterol + cholesterol esters] (WE/[C + CE]), showed a distinct change from infancy through maturity to senescence; the curve of the ratio made a peak in our subjects's 20s. Using the fatty acid analyses, we found an interesting relationship between C16:1 straight and C16:1 iso-branched chains, each of which occupied a large proportion in the fatty acids of wax esters; the former increased in proportion from infancy toward the 20s, with a correlation with aging (r = 0.788, p less than 0.01), and decreased thereafter until our subject's 50s (r = -0.611, p less than 0.01). In contrast, the proportion of the latter followed an entirely reversed course with advancing age. The percentages of C16:1 straight chain components were correlated positively with the WE/[C + CE] ratio (r = 0.642, p less than 0.01), while there was found to be a negative correlation between the proportion of C16:1 iso-branched chain components and the WE/[C + CE] ratio (r = -0.556, p less than 0.01). The results suggest that more active sebaceous glands in lipid production excrete lipids with a higher proportion of C16:1 straight chain fatty acid and a lower proportion of C16:1 iso-branched chain fatty acid. As well as the sebaceous gland activity, the fatty acid composition in sebum wax esters is affected by advancing age in Japanese.

Relationship between cholesterol sulfate and intercellular cohesion of the stratum corneum: demonstration using a push-pull meter and an improved high-performance thin-layer chromatographic separation system of all major stratum corneum lipids.

To investigate the role of cholesterol sulfate (CS) as an intercellular glue or cement in the stratum corneum, we compared the relationship between CS levels and magnitude of the intercellular cohesion of the stratum corneum between the palm and the upper arm. Using a push-pull meter, the palm displayed approximately seven times the magnitude of cohesion of the stratum corneum as the upper arm (n = 11). CS and other stratum corneum lipids were extracted from the palm and the upper arm (n = 22) by a cup method and determined by our improved high-performance thin-layer chromatography (HPTLC). Despite a great difference in the magnitude of cohesion (p less than 0.01), CS levels and ratios of CS to ceramides and CS to cholesterol in the stratum corneum showed no significant differences between the palm and the upper arm. Our results suggest that differences in CS cannot account for the differences in cohesion between palm and upper arm.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation.

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT-4 or U937 cells following X-irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac-DEVD-CHO. p35 was also produced in vitro by treating MOLT-4 cell lysate with recombinant caspases, suggesting that p35 was a caspase-cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA-PKcs. However, p60 was found in ataxia-telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA-PKcs but not ataxia-telangiectasia mutated (ATM).

A single dose of melatonin prevents the phase delay associated with a delayed weekend sleep pattern.

STUDY OBJECTIVES: This study was designed to test the hypotheses that a delayed weekend sleep pattern may lead to a phase delay of the endogenous circadian rhythm, and that melatonin administration can counteract the phase delay and prevent the sleep and functional impairments associated with this sleep pattern. DESIGN: A within-subject, counterbalanced design was used in which each subject participated in both placebo and melatonin conditions. Subjects' sleep-wake schedules were delayed by two hours on Friday and Saturday to simulate the delayed weekend sleep pattern. Six mg of melatonin or a placebo pill was administered double blind on Sunday late afternoon. SETTING: N/A. PARTICIPANTS: Ten healthy volunteers (mean age = 22.1 years old). MEASUREMENTS AND RESULTS: Salivary dim-light melatonin onset (DLMO) was measured on Friday and Monday nights. Subject's sleep was recorded with polysomnography on Sunday night and their levels of sleepiness, cognitive functioning and mood were assessed on Sunday night and Monday morning. Results show that the delayed weekend sleep pattern caused a 31.6 min delay of the endogenous melatonin rhythm. Melatonin administration counteracted the phase delay of endogenous melatonin onset. On Sunday, melatonin administration increased the sleepiness throughout the evening and reduced sleep onset latency at bedtime. On Monday morning, subjective sleepiness was decreased in the melatonin condition. CONCLUSION: A delayed weekend sleep pattern did show a mild phase-delay effect on the endogenous circadian rhythm. A single dose of melatonin can acutely reverse the weekend drift.

Using H2(15)O and C15O in noninvasive pulmonary measurements.

Regional pulmonary blood flow (PBF), regional pulmonary extravascular water (PEW), and regional pulmonary blood volume (PBV) were measured quantitatively with positron emission tomography (PET) using H2(15)O and C15O in normal human subjects and patients before and after thoracotomy. The method was based on the Kety's model and a modification of the Mintun's method of measuring regional PBF with H2(15)O. The method consisted of intravenous injection of H2(15)O and dynamic PET scanning of the lung field and the right heart blood pool. Another scan following C15O inhalation was performed to visualize the blood pool, and the influence of the blood pool radioactivity to the H2(15)O image was subtracted. A mathematic model was applied to the data analysis, and the least-square fitting procedure was used with a computer for obtaining the optimal parameters. The mean values of the measured PBF, PEW, and PBV in two normal human subjects were 67.2 +/- 23 ml/min/100 ml, 17 +/- 5 ml/100 ml, and 19 +/- 6 ml/100 ml, respectively. The PBF, PEW, and PBV increased in the direction of gravity in the transverse cross-section image of the lung. Using this method, we studied the condition of patients who underwent lung surgery with thoracotomy. The PEW increased and the PBF decreased in the patients after surgery. This condition lasted for a few days, and the patients then recovered. The well-known phenomenon of pulmonary edema occurring after thoracotomy, which is usually recognized by symptoms and radiologically, was thus confirmed quantitatively by our method.

Fatty acid composition of sebum wax esters and urinary androgen level in normal human individuals.

To investigate the effects of androgens on the fatty acid compositions of sebum wax esters, we examined sebum and urinary samples from 36 healthy individuals, aged from 3 to 59 years. The percentages of C16:1 straight chain components in wax esters were correlated positively with the urinary testosterone levels in both sexes, and with the urinary levels of etiocholanolone and total 17-ketosteroids (17-KS) in females. These data suggest that more active sebaceous glands in lipid production excrete sebum with a higher proportion of C16:1 straight chain fatty acid, which is considered to be purely endogenous. It appears, therefore, that the proportion of C16:1 straight chain fatty acid in sebum wax esters may indicate the sebaceous gland activity in both sexes. In comparison of the amounts of various straight and terminally branched fatty acids in sebum with urinary androgen levels, the straight even fatty acids tended to change in a positive correlation with testosterone levels, in contrast to the changes of the iso even fatty acids in both sexes and to those of the iso odd fatty acids in males. The straight odd fatty acids showed a similar change to that of the straight even fatty acids in males, while in females, there was no significant correlation between the amounts of the fatty acids and testosterone levels. Anteiso fatty acids showed no notable change correlated with testosterone levels. This result suggests that the synthesis of iso or anteiso fatty acids may be controlled by complex factors and that there may be a unique source of anteiso fatty acids in human sebaceous glands.

Studies on the biosynthesis of cholestanol in cultured cells.

The presence of cholestanol had never been reported in cultured cells, but in the present study cholestanol was detected both in human and rat skin fibroblasts. The present study has shown that the origin of cholestanol in these fibroblasts was derived from de novo synthesis in the cells rather than from the fetal calf serum used in the growth medium. The activity of 5 alpha-cholestan-3-one reductase which is the enzyme catalyzing the final step in the biosynthesis of cholestanol was measured by mass fragmentography using deuterium labeled 5 alpha-cholestan-3-one as a substrate. The activity of this enzyme was also detected in the microsomal fractions of rat cerebrum and cerebellum as well as in the liver. These results suggest that cholestanol can probably be synthesized in extra-hepatic tissues as well as in the liver.

Simplified determination of cholestanol in serum by gas-liquid chromatography: biochemical diagnosis of cerebrotendinous xanthomatosis.

Cholestanol (5 alpha-cholestan-3 beta-ol) in human serum was determined by gas-liquid chromatography after removal by epoxidation of cholesterol which is present in amounts about 400 times those of cholestanol and disturbs the accurate determination of minor sterols in serum. Epicholestanol (5 alpha-cholestan-3 alpha-ol) was used as an internal standard. By this method the cholestanol content in sera of 7 patients with cerebrotendinous xanthomatosis was determined. The cholestanol levels in those sera were significantly higher than those of normal subjects and the present method proved to be useful in a biochemical diagnosis of cerebrotendinous xanthomatosis.

Axonal projection of olfactory sensory neurons during the developmental and regeneration processes.

We have studied the projection of olfactory sensory neurons (OSNs), during the developmental and regeneration processes, using the transgenic mouse carrying the differently tagged odorant receptor genes, MOR28. We have found that the axon terminals of the two sets of MOR28-positive OSNs, one expressing the lacZ tag and the other expressing the green fluorescent protein gene, are dispersed and intermingled at early developmental or regeneration stages. Projection areas become more distinct and separated at later stages, however, two sets of axon fibers are not typically bundled or segregated during pathfinding. It appears that segregation of axons mainly occurs when they target at the olfactory bulb to form the glomerular structure.

Radiotherapy of cholangiocarcinoma: the roles for primary and adjuvant therapies.

A total of 22 patients with cholangiocarcinoma who had been treated with external radiotherapy between 1978 and 1989 were analyzed. Of the 22 patients, 18 had cancer of the hepatic hilus (Klatskin) and 4 had intrahepatic biliary cancer; all but 2 of the subjects had advanced disease. In all, 16 patients underwent primary irradiation for unresectable tumors, 4 were subjected to adjuvant irradiation after gross tumor resection, and 2 received preoperative irradiation followed by gross tumor resection. The mean initial irradiation dose was 52.0 Gy (range, 26-78 Gy). The TDF (time-dose-fractionation) for the entire course of radiotherapy ranged from 49 to 154 (mean, 100). The median survival of all patients was 10 months, and the cumulative 1-year survival value was 37.7%. The external radiotherapy proved to be effective in the treatment of cholangiocarcinoma in terms of palliation and survival.

Intracerebral hemodynamics probed by near infrared spectroscopy in the transition between wakefulness and sleep.

Previous imaging studies have shown that cerebral metabolism is gradually reduced at the beginning of sleep. Few studies have examined the sleep state transition periods from wakefulness to sleep and sleep to wakefulness. The current study used the Near Infrared Spectroscopy (NIRS) technique to describe the intracerebral hemodynamics at the frontal pole in the circumscribed period between wakefulness and sleep. Nine healthy young adults were studied during afternoon naps. Optical probes were placed on the forehead and EEG electrodes on the scalp. At sleep onset oxygenated hemoglobin (oxy-Hb) was reduced (P<0.01) and deoxygenated hemoglobin (deoxy-Hb) showed a near significant reduction (P<0.063). At sleep offset there were increases in oxy-Hb (P<0.005) and deoxy-Hb (P<0.05). In 18 of 26 transitions to sleep there was a coordinated fall in both NIRS parameters, we call the Switch Point, that lasted a mean of 3.6 s. In 32 of 36 transitions to wakefulness there was an analogous Switch Point that lasted a mean of 3.4 s. Before and after the Switch Point, changes were small and the relationship between oxy-Hb and deoxy-Hb was a combination of parallel and reciprocal fluctuations. A synchronized, parallel and short-lived change in oxy-Hb and deoxy-Hb is a discrete event in the transition period between wakefulness and sleep. The concentration of these light absorbing molecules is abruptly set to a new level at sleep-wake transitions and probably reflects the different perfusion demands of these states.

Serum bile acid profiles in cerebrotendinous xanthomatosis.

Non-sulfated bile acid concentrations in sera of 10 cases of cerebrotendinous xanthomatosis (CTX) were determined by mass fragmentography. Total bile acid (TBA) in serum was 0.492 +/- 0.436 microgram/ml (mean +/- SD) which was significantly lower than that (1.481 +/- 0.571) in healthy control sera. Cholic acid was 0.342 +/- 0.291 microgram/ml and was the dominant bile acid, which constituted 69.5% of TBA in serum. Chenodeoxycholic acid was 0.111 +/- 0.133 microgram/ml being a minor component in CTX sera, although it was the major bile acid in healthy control sera. Other bile acids such as deoxycholic acid, lithocholic acid and ursodeoxycholic acid were scarcely detected. Subnormal TBA level and deranged bile acid composition in CTX sera may reflect the defect of bile acid biosynthesis in CTX patients. Determination of serum bile acid may be useful in the diagnosis of CTX.

Involvement of c-Jun NH2-terminal kinase-1 in heat-induced apoptotic cell death of human monoblastic leukaemia U937 cells.

PURPOSE: To determine the involvement of c-Jun NH(2)-terminal kinase-1 (JNK1) and possibly of HSP27 in heat-induced apoptosis of human monoblastic leukaemia U937 cells. MATERIALS AND METHODS: Dominant negative JNK1 (APF), in which the phosphorylation sites Thr-Pro-Tyr were changed to Ala-Pro-Phe, was overexpressed in U937 cells. Cell viability and DNA fragmentation were analysed by the erythrosin-B dye exclusion test and by agarose gel electrophoresis, respectively. Expression of activated caspase-9, phosphorylated JNK1, JNK2, p38 and HSP27 was examined by Western blotting. JNK1 kinase assay was also performed using c-Jun as a substrate. RESULTS: Loss of viability, activated cleavage form of caspase-9 and DNA fragmentation were rapid in U937 cells after 44 degrees C hyperthermia, while overexpression of dominant negative JNK1 interfered with phosphorylation or activation of JNK1 without affecting that of JNK2 or p38/SAPK, and apparently delayed or reduced cleavage and activation of caspase-9, DNA fragmentation and cell death. Heat-induced phosphorylation of HSP27, observed in parental U937 cells, was suppressed and only slightly detectable in jnk1 mutant cells. CONCLUSIONS: Prolonged phosphorylation or activation of JNK1 was considered important for heat-induced apoptosis and JNK1 may control the process possibly through phosphorylation of HSP27 and caspase-9 activation in U937 cells.

Simplified determination of serum cholesterol sulfate by gas-liquid chromatography combined with cyclohexylsilane-bonded phase column purification.

A new gas-liquid chromatographic (GLC) determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) for a biochemical diagnosis of recessive X-linked ichthyosis (RXLI) is described. Although the GLC method for determination of CS is known to be more sensitive than the thin layer chromatographic (TLC) method, the former method has not been widely employed because of its complicated pre-purification steps. The present method allows us to measure the serum levels of CS and DHEAS without tedious purification steps such as multiple conventional column chromatography and preoperative thin layer chromatography. Sulfated steroids are rapidly purified with a commercially available mini disposable cyclohexylsilane-bonded phase (CH) column, CH BOND ELUT, and the purified steroids after desulfation are converted to water-resistant tert-butyldimethylsilyl ether derivative for the GLC analysis on dual 2m glass columns packed with 2% XE-60 on Chromosorb W. By the present method, serum CS concentrations in RXLI patients were shown to be about 10 times higher than those in patients with ichthyosis vulgaris, carriers of RXLI, and healthy subjects. This method is more suitable not only for a biochemical diagnosis of RXLI but also for studies on the metabolism of sulfated steroids than the previous time-consuming GLC methods.

Stratum corneum lipid abnormalities in atopic dermatitis.

Patients with atopic dermatitis (AD) often present with a dry skin. To clarify the relationship between dry skin and lipid abnormalities within stratum corneum, stratum corneum lipids were collected from six AD patients aged 15 to 25 years and from sex- and age-matched controls. All major stratum corneum lipid classes were separated and quantitated by high-performance thin-layer chromatography/photodensitometry. Six ceramide fractions were also isolated and quantitated by thin-layer chromatography/photodensitometry. Esterified fatty acids of both ceramide 1 (acylceramides) and wax esters were analysed by capillary gas chromatography. The relative amounts of all the stratum corneum lipid classes including squalene, cholesterol esters, wax esters, triglycerides, free fatty acids, cholesterol, ceramides, cholesterol sulphate and phospholipids did not differ statistically between AD patients and controls. However, a significant decrease in proportion of ceramide 1, which is believed to be a carrier of linoleate responsible for a water-barrier function, and increased levels of esterified C18:1 fatty acids (oleate) of ceramide 1 were observed in AD patients. On the other hand, the fatty acid compositions as well as the proportions of C16:1 straight-chain component in sebum wax esters of AD patients were very similar to those of controls. These results suggest that a significantly reduced amount and/or structural alterations of ceramide 1 deriving from epidermal keratinocytes may be responsible for the impaired water-barrier function of the skin in AD.