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.

Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls.

A series of novel synthetic monohydroxy polychlorinated biphenyls (OH-PCBs) (5 trichloro-, 5 tetrachloro- and 5 pentachloro-compounds) have been characterized (1H and 13C NMR and high resolution MS) and their estrogenic and thyroid hormone activities assessed using a yeast two-hybrid assay, both with and without possible metabolic activation by rat liver S9 preparation. Moderate estrogenic activity was found for 2,3,4(')-trichlorobiphenyl-4-ol (compound 5) but this was eliminated when exposed to the S9 mix. 2,2('),3('),4,6-Pentachlorobiphenyl-3-ol (13) and 2('),3,3('),6-tetrachlorobiphenyl-4-ol (10) both showed weak estrogenicity in the absence of the S9 mix. The estrogenicity of compound (10) was enhanced 10-fold by exposure to S9 metabolic activation but that of compound (13) remained unchanged. 2('),4,5('),6-Tetrachlorobiphenyl-2-ol (6) showed strong thyroid hormonal activity (5% of that of T4) whereas 3('),4,6-trichlorobiphenyl-3-ol (4), compound (10) and 2,3('),4,5('),6-pentachlorobiphenyl-3-ol (14) showed moderate activity, and 2('),3,3('),5-tetrachlorobiphenyl-2-ol (8) and 3,3('),5,5('),6-pentachlorobiphenyl-2-ol (11) showed weak activity. The activity of (4) was eliminated by S9 metabolic activation whereas those of (6) and (14) were weakened and that of (10) remained unchanged.

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.

Parallel induction of nitric oxide and tetrahydrobiopterin synthesis in alveolar macrophages.

BACKGROUND: Nitric oxide (NO) and an essential cofactor for both constitutive and inducible NO synthase (NOS) activity, tetrahydrobiopterin (6R-L-erythro-1',2'-dihydroxypropyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine; BH4), are thought to be important modulators of function in normal and inflamed airways. However, the exact pathologic roles of NO and BH4 remain obscure. Even less is known about the effects of cytokines on alveolar macrophages. OBJECTIVE: This study was designed to determine whether NO and BH4 are induced by cytokines in mouse alveolar macrophages and to investigate whether NO synthesis is affected by changes in intracellular BH4 levels in alveolar macrophages. METHODS: We compared the induction by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) of NO production and BH4 synthesis in alveolar macrophages. To determine whether NO synthesis is affected by changes in intracellular BH4 levels in alveolar macrophages, we used inhibitors of BH4 biosynthesis. RESULTS: Activation of alveolar macrophages induced parallel increases in NO and intracellular BH4 levels, although induction of the latter appears to be somewhat more sensitive than that of the latter to diverse cytokines. Inducible NO production in alveolar macrophages was blocked by inhibitors of BH4 biosynthesis. IL-2, an important component of the immunomodulatory system, was only a weak activator of alveolar macrophages by itself but potently synergized with IFN-gamma to stimulate the production of both NO and BH4. CONCLUSION: Our results suggest that BH4 synthesis in alveolar macrophages is a potential target for therapeutic intervention in airway inflammatory diseases, such as asthma, cystic fibrosis, and acute bronchial infections whose pathology may be mediated by overproduction of NO.

Water quality characteristics of forest rivers around Lake Biwa.

Forest river surveys were carried out at upper streams of several rivers in the Lake Biwa watershed to understand the water quality characteristics of the rivers, and to find out their relationships with forest features such as geographical, geological and vegetational data. The results showed: (1) Forests have some purification functions for nitrogen and organic matter, but become sources for most of ionic species. (2) Main mineral species in forest rivers are Ca2+, Mg2+ and Na+, HCO3-, CO3(2-), Cl-, SO4(2-) and SiO2. (3) Loading from forests was 0.4-7 kg/km2/d for TN and 0.01-0.3 kg/km2/d for TP. (4) River quality reflects the properties of each forest, and is unique to the place, especially in ionic species such as Ca2+ and Cl-. (5) A cluster analysis successfully categorized ionic components into several groups.

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.

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.

Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurones.

BACKGROUND: We have previously generated transgenic mice carrying the murine odourant receptor gene, MOR28, tagged with lacZ. In this animal, the endogenous MOR28 is differently tagged with GFP. It was found that the transgenic and endogenous MOR28 genes are expressed in a mutually exclusive manner and that the two sets of olfactory sensory neurones (OSNs), each expressing either the transgenic or the endogenous MOR28, project their axons to separate glomeruli. RESULTS: Our fluorescent in situ hybridization (FISH) revealed that the two endogenous alleles of MOR28 are also mutually excluded for their transcriptional activation. Therefore, we studied whether there would be any segregation in the projection of the two subsets of OSNs: one set expressing the paternal and the other expressing the maternal allele. It was found that the OSNs for both alleles shared the same glomerulus for their projection, but the projection targets were segregated within the glomerular structure. CONCLUSION: Two subsets of neurones expressing either the transgenic or the endogenous MOR28 target their axons to two separate glomeruli based on the differences in the genetic backgrounds, nature of tagging, and chromosomal locations. In contrast, neurones expressing a maternal or paternal allele share the same glomeruli, but tend to target to segregated areas within the glomerular structure. The segregation was more prominent with increased differences in the genetic background between the two alleles.

Slowly progressive insulin-dependent diabetes mellitus in an elderly patient with Graves' disease.

We report a case of slowly progressive insulin-dependent diabetes mellitus in an elderly patient with Graves' disease. A 69-year-old man presented with apathetic thyrotoxicosis and weight loss. Laboratory findings indicated insulin-dependent diabetes mellitus (IDDM) with Graves' disease. Human leukocyte antigens DR4 and DR9, which are recognized as markers for IDDM with autoimmune thyroid disease, were detected. The clinical course of the IDDM was compatible with the slowly progressive type. Onset of this disease during old age is rare, and such cases should be analyzed with a thyroid function test because the symptom of thyrotoxicosis may be masked in the elderly.

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).

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.

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.

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.

A case of recessive X-linked ichthyosis: scale-specific abnormalities of lipid composition may explain the pathogenesis of the skin manifestation.

We analyzed the lipid content of the scales, red blood cells, and plasma from a recessive X-linked icthyosis patient. The patient's scales accumulated cholesterol sulfate, had decreased levels of free sterols, sterol esters and sphingolipids, and lacked phospholipids. Although the accumulation of cholesterol sulfate was found in the patient's red blood cells and plasma as well as in the scales, other lipid composition abnormalities were specific for scales. Such scale-specific abnormal lipid composition may explain the pathogenesis of generalized hyperkeratosis and abnormal scaling of the disease.

[Evaluation of image quality of medical facsimile as a radiological image recording system].

We examined the usefulness of a new medical facsimile (MFAX) system in recording and transmitting various kinds of medical images, including X-ray images and colored histopathologic images. The system consists of an image scanner, a magnetic disk for image storage, a transmission circuit and a thermal image printer. Transmission time for a FCR (Fuji computed radiography) image by super-fine mode was 6 minutes. We used ROC (receiver operating characteristics) curves to evaluate the ability of eight radiologists to detect the small simulated nodules placed on an anthropomorphic chest phantom and shown on MFAX images. The radiologists observed both the FCR films and MFAX copies and determined the presence or absence of simulated nodules using five confidence levels. The results obtained for FCR films and MFAX images showed no statistically significant difference.

[Magnetic resonance imaging of renal lymphoma with computed tomography correlation].

The magnetic resonance (MR) imaging and computed tomography (CT) findings in four patients (five kidneys) with non-Hodgkin's lymphoma involving the kidneys and perirenal spaces are presented. The patterns of disease in each case were as follows: bilateral renal nodules, infiltration in the perirenal space, infiltration in the perirenal space with renal involvement, and direct invasion from contiguous retroperitoneum. On plain CT, the lesions showed slight hyperdensity (three kidneys) and isodensity (two kidneys) as compared with normal renal parenchyma. But all lesions appeared as hypodense masses with more definite margins after contrast enhancement. MR imaging findings showed iso- or slight hypointense masses on T1-weighted images and definite hypointense masses on T2-weighted images as compared with the signal intensity of the renal cortex. Dynamic imaging and conventional delayed T1-weighted imaging following Gd-DTPA injection showed no significant enhancement of the lesions. In comparison with contrast enhanced CT, despite its poorer resolution, T2-weighted MR imaging showed nearly the same accuracy in the evaluation of number and extent of the lesions without contrast medium administration. MR imaging was also useful to evaluate the patency of vessel lumen surrounded by tumor mass and to determine the location and extent of huge lesions by its multiplanar imaging capabilities.

Isolation and characterization of gastric trypsin from the microsomal fraction of porcine gastric antral mucosa.

A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in the vesicle fraction. Two forms of the protease were purified over 6,000-fold from the ER-microsome fraction. Analyses of various molecular and enzymatic characteristics including the N-terminal and partial internal amino acid sequences of both forms revealed that they share the same properties and are indistinguishable from porcine pancreatic trypsin. This is the first time that trypsin or a protease almost identical with trypsin has been found to be present intracellularly in normal tissues. The gastric trypsin activities from the ER-microsome and the vesicle fractions were located in distinct density regions upon density gradient centrifugation, which indicates association of the protease with different organelle membranes. Taken together, these results suggest that there may be a novel function of trypsin in the gastric mucosa; it might function as a specific degrading or processing enzyme as an intracellular protease.

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.

Accentuated temporal line on the frontal skull radiograph: a sign of hyperparathyroidism.

PURPOSE: To determine the importance of a temporal line (the marginal line for the attachment of the temporal muscle to the skull) that is accentuated on frontal skull radiographs of hyperparathyroid patients owing to subligamentous bone resorption under the temporal muscle. MATERIALS AND METHODS: Radiographs from skeletal surveys of 134 surgically treated patients with primary (n = 102) or secondary (n = 32) hyperparathyroidism (HPT) and frontal skull radiographs of 63 age-matched control patients were reviewed. RESULTS: An accentuated temporal line was the most frequent finding (29.4% of cases) in primary HPT, followed by subperiosteal bone resorption in the hand (8.6%), salt-and-pepper appearance of the skull (3.5%), and rugger-jersey spine (1.1%). In secondary HPT, an accentuated temporal line became less obvious as subperiosteal bone resorption advanced. This finding was not seen in the control group. CONCLUSION: An accentuated temporal line is another radiographic indication of bone resorption in HPT.