Molecular genetic conspecificity of Spiculopteragia houdemeri (Schwartz, 1926) and S. andreevae (Drózdz, 1965) (Nematoda: Ostertagiinae) from wild ruminants in Japan.

Male dimorphism of the subfamily Ostertagiinae (Nematoda: Trichostrongylidae) is a well-known phenomenon, and two or more morphotypes of a single species have previously been described as different species. Two Spiculopteragia spp., S. houdemeri (syn. S. yamashitai) and S. andreevae (syn. Rinadia andreevae) recorded in Asian cervids and wild bovids, are considered to represent major and minor morphs of S. houdemeri, respectively, based solely on their co-occurrence in the same host individual along with monomorphic females. In this study, males of morph houdemeri ( = S. houdemeri) and morph andreevae ( = S. andreevae) as well as females with three different vulval ornamentations were collected from sika deer (Cervus nippon) and Japanese serows (Capricornis crispus) distributed on the mainland of Japan. Morphologically characterized worms were subjected to molecular genetic analyses based on the internal transcribed spacer region of the ribosomal RNA gene and a partial region of the cytochrome c oxidase subunit I gene of mitochondrial DNA. Of 181 collected sika deer, 177 (97.8%) and 73 (40.3%) deer harboured males of morphs houdemeri and andreevae, respectively. Worm numbers of the former morph were found to range between 1 and 444 per individual, whereas only 1-25 worms per individual were detected for the latter morph. Five out of six serows harboured 47-71 or 2-9 males of morphs houdemeri and andreevae per individual, respectively. Females with one or two vulval flaps were predominant, but there was a substantial presence of flapless females in both host species. All the morphs of male and female adults had an identical genetic background, thus directly confirming the morphological polymorphism of S. houdemeri.

Differential alterations in expressions of ryanodine receptor subtypes in cerebellar cortical neurons of an ataxic mutant, rolling mouse Nagoya.

This study aimed to clarify changes in the spatial expressions of types 1, 2 and 3 ryanodine receptors (RyR1, RyR2 and RyR3) in the cerebellum of a Ca(2+) channel alpha(1A) subunit mutant, rolling mouse Nagoya. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that the mRNA signal levels of RyR1 and RyR3 were altered in the rolling cerebellum, which exhibited lower densities of RyR1 bands and higher densities of RyR3 bands than in the control cerebellum. Quite consistent with the RT-PCR results, the staining intensity of RyR1 and RyR3 was altered in the rolling cerebellum. RyR1 immunostaining appeared in somata and the proximal dendrites of Purkinje cells, and the staining intensity of both subcellular regions was equally lower in all cerebellar lobules of rolling mice than in those of controls. Although RyR3 immunostaining appeared in the dendrites of granule cells, more intense RyR3 staining in rolling mice than in controls was uniformly observed throughout all cerebellar lobules. The present study further examined co-localizations of ryanodine receptor subtypes and voltage-gated Ca(2+) channel alpha(1) subunits in the rolling cerebellum. Somatodendritic RyR1 immunostaining in Purkinje cells overlapped with either a mutated Ca(2+) channel alpha(1A) subunit (P/Q-type), or a Ca(2+) channel alpha(1C) subunit (L-type; dihydropyridine receptor) immunostaining. Immunostaining of these alpha(1) subunits also emerged in granule cells. Those results suggest non-region-related alterations in RyR1 and RyR3 expressions in the rolling mouse cerebellum. Such expressional changes in ryanodine receptor subtypes may be involved in Ca(2+) channel alpha(1A) subunit gene mutation, and may alter regulation of intracellular Ca(2+) concentrations in cerebellar cortical neurons.

Fetal and maternal plasma levels of immunoreactive somatostatin at delivery: evidence for its increase in the umbilical artery and its arterio-venous gradient in the feto-placental circulation.

The concentrations of immunoreactive somatostatin (IR-SRIF) in plasma samples taken from the umbilical artery (UA), umbilical vein (UV), and maternal antecubital vein (MV) were measured in 23 cases of normal delivery at term. High concentrations of IR-SRIF were detected in the plasma from the UA (mean +/- SD, 73.2 +/- 40.6 pg/ml), the value being about 2.5 times that in the UV (29.5 +/- 17.5 pg/ml; P less than 0.001). An arterio-venous gradient was observed in all 23 subjects. The plasma level of IR-SRIF in the MV (10.9 +/- 4.6 pg/ml) was significantly lower than those in the UA (P less than 0.001) and UV (P less than 0.001) and was almost the same as that in nonpregnant women (12.7 +/- 5.4 pg/ml). Chromatographic analysis of an extract of plasma from the UA gave only one peak of immunoreactive material, which was eluted in the same position as synthetic SRIF-14. A similar result was obtained in nonpregnant women. No correlation was found between the plasma level of IR-SRIF and that of GH, insulin, glucagon, or gastrin in the UA. The present findings suggest that the high concentration of IR-SRIF in the feto-placental circulation originates from the fetus and reflects a particular role of this peptide in the process of functional maturation of the neuroendocrine system in early human life.

Radioimmunoassay of an analog of luteinizing hormone-releasing hormone, [D-Ser(tBu)]6des-Gly-NH2(10) ethylamide (Buserelin).

A sensitive and specific radioimmunoassay is described for plasma and urinary levels of [D-Ser(tBu)]6des-Gly-NH2(10) ethylamide (buserelin). No appreciable cross-reaction (less than 0.05%) was observed with LH-RH and its analogs other than buserelin fragments (1.6-45%). The sensitivity was 3 pg per tube. At buserelin concentrations of 125, 250 and 500 pg/ml, the intra- and inter-assay coefficients of variation were 7.9, 10.0 and 10.0%, and 19.0, 7.8 and 6.8% respectively. Recovery of buserelin added to plasma was quantitative (62.5 pg/ml, 101.6%; 125 pg/ml, 76.8% and 250 pg/ml, 63.4%). A dose of 5 micrograms buserelin injected subcutaneously into 5 normal male adults, reached a peak plasma level in 45 min (mean value 119.3 +/- 47.3 pg/ml) and remained detectable for at least 4 h. The half disappearance time was 118.8 +/- 26.0 min. Between 9 and 16% of the administered dose was excreted in the urine within 24 h. Buserelin could also be detected in the plasma after intranasal administration of doses of 150, 300 and 450 micrograms. There was a significant difference in the area under the curve (AUC) for plasma levels after subcutaneous injection of 5 micrograms and intranasal administration of 150 micrograms, but not between the AUC values after the three intranasal doses. These results indicate that this method for radioimmunoassay of buserelin is suitable for analyzing the pharmacokinetics and bioavailability of buserelin in man.

Correlation between permeability-related glycoprotein expression and susceptibilty to oxygen radicals in vincristine-resistant hematologic cell lines.

This study was designed to test the correlation between the expression of permeability-related glycoprotein (P-GP) and susceptibility to oxygen radicals derived from the reaction of hypoxanthine (HX)-xanthine oxidase (XO) in wild type and vincristine (VCR)-resistant hematologic cell lines. A marked correlation between P-GP expression and susceptibility to oxygen radicals was found in VCR-resistant cells, while it was weak in wild cell lines. In contrast, there was neither correlation between sensitivity to VCR and oxygen radicals nor between sensitivity to VCR and P-GP expression in both wild type and VCR-resistant cells. No correlation between sensitivity to adriamycin or oxygen radicals and P-GP expression were observed in both cells tested. These results may suggest a new mechanism of drug resistance in cells expressing P-GP.

Detection of histo-blood group ABO mRNA in human chronic myeloid leukemia cell lines using reverse transcription-polymerase chain reaction (RT-PCR).

ABH carbohydrate antigens are cell surface carbohydrates which occur in three allelic forms, namely A, B and O blood groups. It is unknown how the ABO blood group is expressed in hemopoietic stem cells. In an attempt to verify the ABO mRNA expression in hemopoietic precursor cells, mRNAs were isolated from human chronic myeloid leukemia (CML) cell lines which are believed to be at the most immature level of hemopoietic differentiation among hemopoietic malignancies. In particular, K-562 and KOPM-28 cells were used with the reverse transcription-polymerase chain reaction (RT-PCR) technique for amplifying ABO gene transcripts. The amplified ABO cDNAs from two cell lines were characterized by the digestion of Kpn-I restriction enzyme. The blood types were determined by polymerase chain reaction of the specific allele (PASA) method. Both of the human chronic myeloid leukemia cell lines expressed ABO mRNA. The quantity of ABO mRNA in the K-562 cell line is significantly higher than that of the KOPM-28 cell line. The ABO blood type of these two cell lines was type O. Because the CML cell lines are presumed to be at the immature stem cell level of hematopoietic cell differentiation and because it is believed that the cultured cell lines from hematologic malignancy reflect the characteristics of normal corresponding hemopoietic cells, the hemopoietic stem cells should express mRNA of the ABO blood group.

Effect of MDR antagonists on the cidal activity of vincristine for cells expressing MDR-1 is superior to those expressing MRP.

In an attempt to identify the target protein, P-GP or mrp, of each MDR antagonist, verapamyl (Ver), dipyridamole (Dip), or cyclosporin A (Cy-A), this study was designed to compare the activity of the three afore-mentioned drugs and to test their combined effect on the cidal activity of vincristine (VCR) in five types of wild and the corresponding VCR-resistant cultured cell lines from human leukemia and lymphoma. Three of the VCR-resistant cell lines are characterized by the overexpression of mdr-1, while two cell lines overexpress mrp. We found that all three antagonists additively to synergistically enhanced the cidal activity of VCR for the five wild-type and VCR-resistant cell lines in a dose dependent manner when used singly. Combinations consisting of a 20% inhibitory concentration (IC20) of VCR plus two antagonists also showed additive to synergistic effects on both wild and VCR-resistant cell lines. It is of interest that the combined effect of IC20 VCR plus MDR antagonists on the three VCR-resistant cell lines expressing mdr-1 was significantly superior to those of the two cell lines expressing the mrp gene. These results suggest that the combined effect of MDR antagonists work better than their single use and that the MDR antagonists work more efficiently in cells showing drug resistance through mdr-1 than in those utilizing mrp.

Co-storage and co-secretion of somatostatin and catecholamine in bovine adrenal medulla.

Co-storage and co-secretion of somatostatin (SRIF) and catecholamine (CA) were demonstrated using bovine adrenal medulla. In the experiment of retrograde perfusion system of isolated adrenal gland, the basal concentration of immunoreactive (IR)-SRIF was 20 pg/4 ml/min, but a significant increase of the value (142 pg/4 ml/min) was observed at 1-2 min after a brief infusion of acetylcholine (10 mM) and the marked release continued for over 6 min. Similar result was obtained for CA release. On gel-filtration chromatography of a soluble lysate of chromaffin vesicles prepared by differential and discontinuous sucrose density centrifugation, IR-SRIF was separated into three components with molecular sizes corresponding to prepro-SRIF (76.8% of the total immunoreactivity), SRIF28 (14.1%) and SRIF14 (9.1%).

Overcoming of cross-resistance to the cell-killing activity of oxygen radicals in VCR-resistant hematologic cell lines with cyclosporin-A.

Anti-cancer agents like adriamycin, mitomycin-C, bleomycin, and etoposide express their cell-killing activity partly through oxygen radicals. Since vincristine (VCR)-resistant cells show cross-resistance to oxygen radicals, this study was designed to study whether the combined effects of oxygen radicals derived from hypoxanthine-xanthine oxidase plus enhancers of anti-cancer drugs such as verapamil (Ver), dipyridamole (Dip), and cyclosporin-A (Cy-A) overcome the cross-resistance for oxygen radicals in VCR-resistant human leukemia/lymphoma cell lines. We found that 3 micrograms/mL Cy-A enhances the cidal effect of oxygen radicals on two of five types of wild type and two of five VCR-resistant cell lines and it could overcome the cross-resistance to oxygen radicals in VCR-resistant cells. Neither Ver nor Dip showed any effect on the cidal activity of oxygen radicals. Thus, it was suggested that Cy-A works with different mechanisms from those of Ver or Dip, and Cy-A may be useful in reducing the doses of anti-neoplastic agents that exert their activity via oxygen radicals in the clinical treatment.

Subclinical pseudohypoparathyroidism type II: evidence for failure of physiologic adjustment in calcium metabolism during pregnancy.

Patients with latent disorders of hormone response mechanism are rarely found. This paper reports a case of subclinical pseudohypoparathyroidism type II in which physiological adjustment of calcium (Ca) metabolism became insufficient only in the second half of pregnancy. A 34-year-old woman examined for a slight bruise on the head was incidentally found to have marked intracranial calcification and a full set of false teeth. From her history of past pregnancy, it was revealed that she suffered from symptoms of hypocalcemia during late gestation (serum total Ca level, 4.8-6.4 mg/dl), which disappeared spontaneously after delivery. When the woman was not pregnant, although only the total Ca level was slightly below the normal range (7.7-8.4 mg/dl), the serum ionized Ca, phosphorus (P), magnesium, 1,25-dihydroxycholecalciferol and 24,25-hydroxycholecalciferol levels, plasma parathyroid hormone (PTH) level and urinary excretion of Ca were all normal without treatment. Intravenous infusion of 30 mg/kg EDTA-2Na resulted in marked elevation of plasma PTH associated with significant reduction of serum ionized Ca. In contrast, although her urinary excretion of phosphorous per hour was within the normal range in the basal state, she showed no proportional change in urinary phosphorous excretion with increase in urine cyclic AMP induced by administration of PTH(1-34). From these findings, she was diagnosed as having an incomplete form of pseudohypoparathyroidism Type II. This abnormality seems to be rare, but we consider that the present observations provide important information for preventive care of pregnant women and fetuses during gestation.

Takayasu's arteritis associated with antiphospholipid antibodies. Report of two cases.

The authors describe 2 patients with Takayasu's arteritis in whom lupus anticoagulant was positive and the titer of anticardiolipin antibody was elevated. One patient developed diffusely stenotic and occlusive changes in the multiple larger arteries. Histology of the small-sized arteries in another patient showed occlusive vasculitis without thrombosis, in addition to the findings in large-sized arteries compatible with Takayasu's disease. These findings are uncommon in Takayasu's arteritis. These findings suggest that antiphospholipid antibodies may have contributed to the pathogenesis of the extensive vasculopathy and may have triggered vasculitis in these patients.

[Genetic analysis of the genotype of ABO blood group with the DNA from a hair].

The genotype of the hair genomic DNA from 14 subjects with the ABO blood group was determined using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. The amino acid substitutions of codon 87 and 176 of ABO allelic cDNAs were analyzed to distinguish A, B, and O alleles by restriction enzyme digestion. To identify codon 87, the 249bp DNA fragment was amplified by PCR and digested with Kpn I. To identify codon 176, the 285bp DNA fragment was amplified by PCR and digested with Ban I. The genotype of the 14 ABO type-known subjects could be identified by the analysis of the digested DNA fragments. These findings indicate the usefulness of the PCR-RFLP method for determining the ABO genotype with the DNA from only one hair.

[Direct determination of ABO and cisAB blood group genotypes using polymerase chain reaction amplification of specific alleles (PASA)--method].

The genotypes of genomic DNAs of 20 normal subjects with ABO blood group and 12 subjects with cisAB blood group were directly determined using polymerase chain reaction (PCR) amplification of specific alleles (PASA)-method. This method is based on the fact that PCR amplification occurs only when the 3' endbase of the primer is matched to the nucleotide of No. 261, 526, 796 or 803 of ABO allelic cDNA. And three of five regions of allelic DNAs are co-amplified in a single PCR (multiplex-PCR) in this study. ABO and cisAB blood group genotypes are directly determined, based on the molecular size of allele specific amplification products that contain 261, 526, 796 and 803 nucleotide (the sites of amino acid substitutions). The method requires only about 4 hours from starting up of PCR to the results, so it is rapid, simple and useful for detecting the genotype of ABO and cisAB blood groups.

[Genetic analyses of the ABO blood groups and application of the clinical laboratories].

Gene technology using polymerase chain reaction (PCR) has markedly advanced in recent year and has been introduced in clinical laboratories. In this paper, the genotypes of genomic DNAs of subjects with cisAB blood group were analysed using three methods, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and the PCR-direct sequencing method, and directly determined using the polymerase chain reaction (PCR) amplification of specific alleles (PASA)-method. The differences among the methods were as follows, PCR-RFLP and PCR-direct sequencing method require 2-step procedures, and are complicated for clinical laboratories. The PASA method is based on the fact that PCR amplification occurs only when the 3' endbase of the primer is matched to sites of the nucleotide substitution of ABO allelic cDNA. Three of five regions of allelic DNAs were co-amplified in a single PCR (multiplex-PCR) in this study. ABO and cisAB blood group genotypes were directly determined, based on the molecular size of allele-specific amplification products. The PASA method requires only about 4 hours from starting PCR to results, making it rapid, simple and useful for detecting the genotype of ABO and cisAB blood groups in comparison with PCR-RFLP and the direct sequencing methods and will allow this procedure to be very versatile and widely used throughout the research and clinical diagnostic communities. The analyses of the nucleotide sequence at nucleotides No. 261, 526, 703, 796 and 803 in 3 major subjects in the cisAB blood group (cisA2B3, cisA1B3 and cisA2B) revealed chimeric structures of the A allele and B allele on the same gene.

[Genetic analyses of the genotypes of ABO and cisAB blood groups].

The genotypes of genomic DNAs of 20 normal subjects with ABO blood group and 12 subjects with cisAB blood group were analysed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and a combination of PCR-RFLP and direct sequencing method, respectively. To identify the codon 87, 249 bp DNA fragment was amplified by PCR and digested with Bst EII and Kpn I. To identify the codon 176, 506 bp DNA fragment was amplified by PCR and digested with Bss HII and Ban I. To identify the codon 235, 266 and 268, 506 bp DNA fragment was amplified by PCR and determined by direct sequencing. Analyses of the digested DNA fragments of codon 87 and 176 in 20 normal subjects with ABO blood group revealed the ratio of homotype (AA, 20%; BB, 20%) and heterotype (AO, 80%; BO, 80%) in subjects with phenotype A or B. Same results were obtained in subjects with phenotype AB or O. The analyses of nucleotide sequence at codons 87, 176, 235, 266 and 268 in 12 subjects with cisAB blood group (6 cisA2B3, 3 cisA2 B and 3 cisA1B3) revealed chimera structure of A allele and B allele on the same gene. These results indicate the usefulness of PCR-RFLP method for determining the genotype of A and B bloods group, and a combination of PCR-RFLP and direct sequencing for determining the genotype of cisAB blood group.

[The right chest electrocardiogram in normal subjects].

Right chest electrocardiograms (ECGs) of 75 healthy subjects (19 men and 56 women; age 18 to 25 years) were recorded in order to study characteristic features of right precordial ST-T and QRS waves. The diagnostic criteria for right ventricular infarction (RVI) by ECG were also tested in healthy subjects to reascertain their value for diagnosing RVI. 1) The QRS configuration in right chest leads was usually the rS pattern in normal subjects (95% of V3R and 80% of V4R). 2) The Q wave was found in 5% of V4R, 20% of V5R and 51% of V6R. 3) ST elevation of 0.05-0.1 mV at 40 ms and 80 ms after the end of QRS deflection was found in 16 and 28% of V3R, 3 and 4% of V4R, respectively. 4) The T waves were usually negative in all right chest leads (79-88%). 5) The Q waves in V5R and V6R and ST elevation in V3R were relatively frequent findings in normal subjects. Therefore, the presence of a Q wave or ST elevation in these leads are not necessarily specific indicators of RVI by ECG. Furthermore, our data revealed that ST elevation with a Q wave in right chest leads was not present in any of the healthy subjects. This finding may be a more specific indicator for the diagnosis of RVI by ECG.

Effect of electric stimulation of the celiac vagus on gastric acid secretion and plasma concentrations of somatostatin and gastrin in the portal and gastroepiploic veins of dogs.

Electric stimulation (ES) of the celiac vagus during tetragastrin infusion reduced significantly the portal plasma concentration of somatostatin (SS) from 113 +/- 11.3 pg/ml to 87.8 +/- 5.8 pg/ml (P less than 0.05) in anesthetized dogs, in parallel with marked decrease of gastric acid secretion (59.9 +/- 7.1% of the prestimulatory value; P less than 0.01). A similar change in the portal plasma SS concentration was observed by ES of the celiac vagus on infusion of saline, with a concomitant significant increase in the portal plasma level of gastrin from a basal value of 65.9 +/- 7.0 pg/ml to a peak value of 129 +/- 29.9 pg/ml (P less than 0.05). However, no fluctuation of the plasma SS or gastrin level in the gastroepiploic vein was detected during or after ES of the celiac vagus. These findings indicate that gastric SS and gastrin are not of primary importance in the mechanism of inhibition of gastric acid secretion induced by ES of the celiac vagus in the dog.

Secretory profile of immunoreactive growth hormone-releasing hormone (IR-GHRH) during sleep in man and its clinical value.

To clarify the role of growth hormone-releasing hormone (GHRH) in the regulation of the episodic growth hormone (GH) secretion which is known to occur constantly in the initial slow wave stage of nocturnal sleep in man, we studied the relation between the secretions of plasma immunoreactive(IR)-GHRH and GH while recording electroencephalograms. In subjects who showed a normal sleep pattern, the plasma IR-GHRH level increased 3- to 4-fold just before the surge of plasma GH, suggesting that GH release in the initial slow wave stage of sleep is mainly mediated by GHRH. However, when there was an apparent GH surge just before the onset of sleep, the magnitude of the GH response associated with the initial slow wave stage tended to be blunted, even when sufficient IR-GHRH was released. We also observed no appreciable fluctuations of plasma IR-GHRH during nocturnal sleep in a patient diagnosed as having GH-deficient dwarfism, suggesting the primary lesion was on the hypothalamus level, not the pituitary, in such a patient. In a case of multiple endocrine neoplasia (MEN) type I with an ectopic (GHRH-producing pancreatic tumor, no remarkable elevation of plasma IR-GHRH was detected in the initial slow wave stage of nocturnal sleep. We conclude that the present study is significant not only in demonstrating the physiology of GHRH release, but also in establishing a safe, reliable and practical test for routine clinical use to investigate intrinsic ability to release GHRH and the primary lesions in patients with disorders of GH secretion.

Analysis of the Gs alpha gene in growth hormone-secreting pituitary adenomas by the polymerase chain reaction-direct sequencing method using paraffin-embedded tissues.

We investigated the prevalence of Gs alpha gene mutations in growth hormone (GH) secreting pituitary adenomas from Japanese patients with acromegaly. Forty-five GH-secreting adenomas were examined for the presence of point mutations in codons 201 or 227 of the Gs alpha gene using the polymerase chain reaction-direct sequencing method and deoxyribonucleic acid extracted from paraffin-embedded tumor specimens. Mutation of codon 227 of the Gs alpha gene was not observed in any of the tumors, but a mis-sense mutation of codon 201 was identified in two tumors (4.4%). One lesion was a densely granulated GH cell adenoma in a patient with adenomatous goiter and breast cancer. The other was a mixed GH cell-prolactin cell adenoma in a patient with multiple endocrine neoplasia type 1 associated with parathyroid hyperplasia and a pancreatic islet cell tumor. The Gs alpha gene detected in parathyroid tissue and pancreatic tumor tissue was of the wild type in this second patient, and the mutation was specific to the pituitary tumor. These results suggest that point mutations of codons 201 or 227 of the Gs alpha gene may not be important mediators of oncogenesis for GH-secreting pituitary adenomas in Japan.

A pituitary specific point mutation of codon 201 of the Gs alpha gene in a pituitary adenoma of a patient with multiple endocrine neoplasia (MEN) type 1.

The DNA from a pituitary adenoma of a patient with multiple endocrine neoplasia (MEN) type 1 was analyzed to detect a point mutation of the Gs alpha gene (gsp) by the PCR direct-sequencing method. The patient had galactorrhea, amenorrhea and acromegalic features. Hormonal examination revealed high serum levels of PRL and GH. The tumor was histologically diagnosed as a mixed GH cell-PRL cell adenoma in which GH and PRL were produced by different cells. Sequence analysis of the DNAs extracted from paraffin sections of pituitary, parathyroid, and pancreas tumors demonstrated the substitution of thymidine for cytidine in codon 201 of the Gs alpha gene that resulted in replacement of arginine (CGT) with cysteine (TGT) only in the pituitary adenoma, but not in the parathyroid and pancreas tumors. These results suggest that a pituitary specific point mutational activation of the Gs alpha gene may be involved in the development of the pituitary adenoma in this patient.