Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies.

Mitochondrial dysfunction observed in situ in cardiomyocytes of rats in experimental diabetes.

OBJECTIVE: The aim was to investigate effects of experimental diabetes and insulin treatment on heart myocytes, particularly on the mitochondrial function studied in situ in isolated cardiomyocytes. METHODS: 20 male Sprague-Dawley rats (140-160 g) were made diabetic by intraperitoneal streptozotocin, 70 mg.kg-1. Ten then received daily subcutaneous injections of ultra lente insulin (starting dose of 3 units.d-1) for 7-15 d from the 20th day after streptozotocin. There was a control group of 11 rats. The rats were killed 21-35 d after the induction of diabetes, and heart myocytes were isolated by collagenase digestion. The 45[Ca]2+ uptake of mitochondria in situ in permeabilised myocytes, the transmembrane potential gradient of mitochondria, and the respiration of myocytes, as well as the cell yield and cell [45Ca]2+ uptake, were examined. RESULTS: Mitochondrial uptake of [45Ca]2+ was significantly decreased in the diabetic group compared to control at cytosolic calcium concentrations between 760 nM and 44.6 microM. The mitochondrial potential of diabetic myocytes, estimated from the distribution of [3H]triphenylmethylphosphonium+, was slightly but significantly decreased from the control value. Cell respiration, measured polarographically in the presence of pyruvate and malate or succinate as oxidisable substrates, and with or without 2,4-dinitrophenol, was decreased by diabetes. The rapidly exchangeable [45Ca]2+ content in the myocyte with intact sarcolemmal membrane ("cell Ca2+ uptake") and the yield of cells from heart tissue were also diminished in diabetic rats. These changes were returned to normal by insulin treatment of 7 d or longer. CONCLUSIONS: Insulin deficiency at early stages causes defects of mitochondrial function detectable in situ in cardiomyocytes. This suggests the possibility that such alterations are causative factors in the development of diabetic cardiomyopathy.

Seasonal variation of serum thyrotropin concentration and thyrotropin response to thyrotropin-releasing hormone in patients with primary hypothyroidism on constant replacement dosage of thyroxine.

Ten patients with primary hypothyroidism (aged 32--66 yr), replaced on constant daily doses of L-T4 (mean +/- SD, 1.90 +/- 0.22 micrograms/kg BW), were used to examine seasonal variations in serum levels of thyroid-related hormones for a period of 14 months. Basal and peak TSH concentrations after TRH (500 micrograms) were higher in winter than in summer. Summer values for basal TSH were all normal (normal range, less than 4.8 microU/ml), while winter values were supranormal in 5 of 10 patients. Summer values for peak TSH were subnormal or normal (normal range, 5.0--40.0 microU/ml), while winter values were supranormal in 3 patients, with the remaining values being normal [log basal TSH, 0.511 +/- 0.438 vs. 0.084 +/- 0.244 (P less than 0.05); log peak TSH, 1.394 +/- 0.410 vs. 1.017 +/- 0.423 (P less than 0.05)]. Serum resin T3 uptake, T4, free T4 index(FT4I), T3, free T3 index, and rT3 levels did not vary seasonally, although T4 and FT4I tended to fall in the winter. The summer and winter QKd interval (the interval from the onset of a QRS complex in the electrocardiogram to the appearance of the Korotkoff sound at diastolic pressure), basal metabolic rate, and serum cholesterol concentrations were all within the normal range. Basal and peak TSH after TRH were inversely correlated with serum T4 and FT4I levels. The basal TSH concentration was further inversely correlated with the seasonally altering ambient temperature. These results indicate that during the treatment of primary hypothyroidism with constant doses of T4, 1) serum TSH and its response to TRH show seasonal variation, 2) the hypersecretion TSH in the winter is related to small changes in serum T4 and FT4I levels, and 3) the seasonal variation in the serum TSH concentration may need to be taken into consideration when evaluating the adequacy of a T4 replacement dose.

Association between dietary iodine intake and prevalence of subclinical hypothyroidism in the coastal regions of Japan.

The prevalence of thyroid dysfunction in relation to iodine intake was studied in adults (n = 1061) in five coastal areas of Japan that produce iodine-rich seaweed (kelp). The prevalence of hyperthyroidism (TSH < 0.15 mU/L) was similar in these areas, whereas that of hypothyroidism (TSH > 5.0 mU/L) varied from 0-9.7%. The relative frequency of above normal iodide concentration in the morning urine (> or = 75 mumol/L) [high urinary iodide (UI)] varied from 3.7%-30.3%. Together with previously reported results of a noncoastal city, the frequency of high UI correlated significantly with that of hypothyroidism with negative thyroid autoantibody (r = 0.829, n = 6, P < 0.05) but not with positive thyroid autoantibody (r = 0.278, NS) or with that of hyperthyroidism (r = 0.038, NS). Hypothyroidism was more prevalent in thyroid autoantibody-negative subjects with high UI (group II, 12.1%) than with normal UI (group I, 2.3%) (P < 0.001). The TSH [21.9(6.5-73.7)mU/L] (mean +/- SD) and thyroglobulin [288 (182-456) micrograms/L] levels in group II were significantly higher than the respective levels in group I [9.6(3.7-25.3)mU/L and 123 (38-399) micrograms/L] (P < 0.05). Free T4 of group II (9.9 +/- 3.9 pmol/L) was significantly lower than in group I (14.2 +/- 3.9 pmol/L) (P < 0.05). These results indicate that 1) the prevalence of hypothyroidism in iodine sufficient areas may be associated with the amount of iodine ingested; 2) hypothyroidism is more prevalent and marked in subjects consuming further excessive amounts of iodine; and 3) excessive intake of iodine should be considered an etiology of hypothyroidism in addition to chronic thyroiditis in these areas.

Characterization of high-affinity binding sites for diisopropylfluorophosphate (DFP) from chicken spinal cord membranes.

The delayed neurotoxic organophosphate [3H]diisopropylfluorophosphate ([3H]DFP) binds with high affinity to membrane-bound proteins from the chicken spinal cord. The DFP binding proteins were solubilized from membrane preparations, using a detergent (CHAPS). The protein(s) sites that labeled with a low concentration of [3H]DFP, e.g. 10(-10)-10(-9) M, were defined as the high-affinity binding sites. The density (or concentration) of the high-affinity binding sites in protein(s) was determined by the difference between total and non-specific binding. The high-affinity binding sites were saturable, and the maximal amount of binding sites was estimated at 400 fmol/mg protein. [3H]DFP binding to solubilized proteins was not completely reversible. Concentration-dependent curves suggested that the [3H]DFP binding sites differ from the active sites of acetylcholinesterase, butyrylcholinesterase, and neuropathy target esterase, as well as from muscarinic acetylcholine receptors. The amount of DFP binding sites after a neurotoxic dose of tri-o-cresyl phosphate (TOCP) decreased markedly in membrane preparations from the chicken spinal cord. These results indicate that a TOCP metabolite(s) interacts with the DFP binding sites in vivo. Gel filtration chromatography of the solubilized membranes indicated at least two major high-affinity DFP binding proteins with apparent molecular weights of 300 and 110 kDa. The DFP binding sites corresponding to the 110 kDa protein were insensitive to eserine, a potent anti-cholinesterase agent.

The depressor function of biceps on the head of the humerus in shoulders with tears of the rotator cuff.

We investigated the function of biceps in 18 patients (19 shoulders) with lesions of the rotator cuff. Their mean age was 59 years. Another series of 18 patients (19 shoulders) with normal rotator cuffs as seen on MRI acted as a control group. Their mean age was 55 years. A brace was used to maintain contraction of biceps during elevation. Anteroposterior radiographs were obtained with the arm elevated at 0 degrees , 45 degrees and 90 degrees with and without contraction of biceps. The distance between the centre of the head of the humerus and the glenoid was compared in the two groups. We found that in the group with tears there was significantly greater proximal migration of the head of the humerus at 0 degrees and 45 degrees of elevation without contraction of biceps but depression of the head of the humerus at 0 degrees, 45 degrees and 90 degrees when biceps was functioning. We conclude that biceps is an active depressor of the head of the humerus in shoulders with lesions of the rotator cuff.

Treatment of hypoglossia-hypodactyly syndrome without extremeity anomalies.

Three cases of hypoglossia-hypodactyly syndrome without limb deformities are reported. All exhibited different degrees of tongue hypoplasia, micrognathia, retrognathia with a very narrow space between the left and right halves of the mandible, constricted isthmus, and only one lower incisor. Bone lengthening for the midline mandibular hypoplasia and orthodontic treatment were performed in the three cases with satisfactory results.

Measurements of serum-free thyroid hormone concentrations by ultrafiltration--a comparison with equilibrium dialysis and mathematical calculation.

An ultrafiltration method (UF) for measuring free thyroxine (FT4) and free triiodothyronine (FT3) using the Diaflow YM membrane (Centricon-10) is described. The results are compared with those by equilibrium dialysis (ED) and also by mathematical calculations derived from T4, T3, and binding protein concentrations. The precision with the UF method was excellent. The normal ranges of FT4 and FT3 by the three methods are all comparable. There was a high degree of correlation of FT4 or FT3 results by UF with those by ED and by calculation (r = 0.940-0.974, n = 161, P less than 0.001). FT4 and FT3 by all methods agreed well for hyperthyroidism, hypothyroidism, and for patients with low T4-binding globulin. The mean FT3 in pregnancy was lower than the normal value for all methods, and FT4 concentrations by UF and calculation also decreased in late pregnancy. The mean FT4 by UF and ED in low T3 syndrome were significantly higher than in the normal controls, while the calculated FT4 was lower. The FT3 in low T3 syndrome distributed normal to subnormal in all methods. These results indicate that a) the UF method is a reliable reference method for measuring FT4 and FT3 concentrations; b) the UF results agree well with those by ED and also with theoretically derived values in subjects with thyroid diseases and TBG abnormalities; c) for patients with low T3 syndrome, the FT4 results obtained by UF and ED are similarly discrepant from the calculated results, implying the existence of binding inhibitor(s) which affect both UF and ED measurements.

The effects of drug metabolism inducers on the delayed neurotoxicity and disposition of tri-o-cresyl phosphate in hens following a single intravenous administration.

Intravenous (iv) administration of tri-o-cresyl phosphate (TOCP) caused a delayed neurotoxic effect in hens similar to that produced by oral and dermal administration. The iv ED50s for producing ataxia and paralysis were estimated to be 15.9 mg/kg and 31.7 mg/kg respectively. The tissue disposition of unaltered TOCP was determined in hens following a single iv injection of 40 mg/kg of TOCP. One hour (hr) after the injection, the leg muscle contained the highest concentration, 26.99 micrograms/g fresh weight followed by the adipose tissue. Among the nerve tissues, the sciatic nerve had the highest concentration, 9.63 micrograms/g followed by the spinal cord and the brain. Except the adipose tissue and the sciatic nerve, the concentration in all analyzed tissues dropped below 1.0 microgram/g (ml) after 24 hr. An unidentified metabolite appeared in bile taken 1 and 3 hr after the injection. Pretreatment of hens with 3-methylcolathrene (3-MC) and beta-naphthoflavone (B-NF) protected against the TOCP-induced delayed neurotoxicity, whereas phenobarbital (PB) failed to protect against the neurotoxicity. Plasma creatine phosphokinase (CK) activity in paralytic birds increased approximately 4 times of the control or symptomless hens on the 21st day. 3-MC-, B-NF- and PB-treatment depressed substantially the concentration of unaltered TOCP in brain and plasma 1 hr after iv dosing with 40 mg/kg of TOCP. Only B-NF pretreatment lowered the level of TOCP in spinal cord. There was no effect of these inducers on the level of TOCP in sciatic nerve and adipose tissue. B-NF and 3-MC lowered significantly the TOCP level in leg muscle, whereas PB had no such effect. More attention should be paid to the role of TOCP in muscle, especially to the leg muscle, judging from the present toxicological and metabolic studies.

Delayed neurotoxicity and toxicokinetics of leptophos in hens given repeatedly by low-dose intravenous injections.

The repeated intravenous injections (RIVInj) of 5 mg/kg/day leptophos [O-(4-bromo-2, 5-dichlorophenyl) O-methyl phenylphosphonothioate] for 3 consecutive days caused delayed ataxia in 4 out of 9 hens (44.4%). And one out of 9 hens (11.1%) given RIVInj of 3 mg/kg leptophos for 5 days was affected with ataxia. Twenty hens, however, which received a single intravenous injection (SIVInj) of 15 mg/kg leptophos did not exhibit any delayed neuropathic signs at all. Thus, delayed neurotoxicity was increased by the subdividing RIVInj of the critical dose which was shown in the SIVInj of leptophos. The leptophos concentration in plasma and liver decreased very rapidly after finish of either SIVInj or RIVInj. Although no significant differences were observed in the biological half life of leptophos in plasma by different dosages, the mean level of leptophos decreased significantly with frequency of injections. On the contrary, the evident accumulation of leptophos was observed in only sciatic nerve with RIVInj. Leg muscle maintained relatively high level of leptophos after the last injection. These results suggest that leptophos seems to transfer from blood to affinitive tissues such as sciatic nerve or leg muscles and to accumulate there easily in initial stage after repeated iv injections, and that this causes the enhancement of neuropathy with repeated administrations of divided critical dose of leptophos in both iv and oral administration.

[A comparison of measurements of serum free thyroxine concentration by equilibrium dialysis and ultrafiltration using undiluted serum].

We have examined the clinical usefulness for free thyroxine (FT4) measurement by equilibrium dialysis of undiluted serum and by radio-immunoassay of T4 concentration in the dialysate (ED/RIA). The results were compared with those by ultrafiltration of undiluted serum (UF/RIA), which is believed to offer the best possible approach towards estimation of in vitro serum level of FT4. The precision with both methods was excellent. There was a high degree of correlation of FT4 concentration by both methods (r = 0.831, p less than 0.001, n = 99). FT4 concentration by ED/RIA and UF/RIA methods agreed well for hyperthyroidism, hypothyroidism, normal pregnancy, and for low T4-binding globulin concentration. In nonthyroidal illness (NTI), FT4 levels were all within or above normal for UF/ED method, while some FT4 values in low T4 syndrome were below normal for ED/RIA method. The effect of progressive serum dilution on FT4 concentration was also examined for both methods. There was no fall in FT4 concentration in sera from normal, hyperthyroid patient and in serum with low T4 syndrome for both methods, but in a serum from low T3 syndrome, there was a fall in FT4 concentration by dilution with UF/RIA method, indicating that serum dilution with ED/RIA method might underestimate FT4 concentration in some NTI sera. From these results it is concluded that the present ED/RIA method is adaptable to clinical laboratory use as a reference standard method for FT4 measurement.

[Normality of serum thyroid hormones, thyroxine binding globulin and thyroid stimulating hormone concentrations in normal adult men (author's transl)].

The normality of serum thyroxine (T4), triiodothyronine (T3), reverse T3, thyroxine binding globulin (TBG) and thyroid stimulating hormone (TSH) concentrations were investigated by using normal probability paper. 107 healthy adult men (age 35-48) were carefully selected; their healthiness was evaluated by physical, biochemical and roentogenologic examinations as well as by intensive interview on their past history. All assays were simultaneously carried out. When each cumulative distribution was plotted on normal probability paper, the straight line was obtained in T4, T3 and TBG but in rT3 and TSH, the lineality was seen only when logarithmic normal probability paper was used. The normal ranges by conventional statistical calculation, i.e., mean +/-2 S.D. was compared with those obtained on normal probability paper. The normal ranges of T4, T3 and TBG agreed well each other, whereas in rT3 and TSH, normal ranges obtained on the probability paper shifted toward higher levels than those by statistical calculations. Thus, it is suggested that the normal ranges of rT3 and TSH should be defined either by statistical calculation of each result after logarithmic transformation or by using logarithmic probability paper.

[Evaluation of fine needle aspiration cytology in the diagnosis of thyroid diseases].

The role of fine needle aspiration cytology (FNA) was examined in 384 patients with thyroid diseases referred to the thyroid clinic from May 1984 to June 1988. The cytological diagnoses were 65 (16.9%) malignant neoplasms, 137 (35.7%) benign neoplasms, 135 (35.2%) chronic thyroiditis, 20 (5.2%) thyroid cysts, and 27 (7.0%) other thyroid diseases including Graves' disease and subacute thyroiditis. Ninety-eight patients were selected for thyroidectomy based on criteria, which included clinical and cytological diagnosis, and the following pathological diagnoses of resected specimens were obtained: 45 (45.9%) papillary carcinoma, 9 (9.2%) follicular carcinoma, 26 (25.5%) follicular adenoma, 1 (1.0%) papillary adenoma, 14 (14.3%) adenomatous goiter, and 3 (3.1%) Hürthle cell adenoma. The accuracy of diagnosis of goiter by FNA was examined by comparing with that by histological findings. False negative rate, false positive rate, true positive rate, and true negative rate of FNA were 5.7%, 10.0%, 94.3%, and 90.0% respectively. The calculated sensitivity, specificity, and accuracy of FNA were 92.6%, 92.3% and 92.5% respectively. These results indicate that the FNA is a safe and reliable method for routine use in the evaluation of nodular thyroid disease.

[Screening for thyroid dysfunction in adults residing in Hokkaido Japan: in relation to urinary iodide concentration and thyroid autoantibodies].

The prevalence of thyroid dysfunction and its relation to thyroid autoantibodies (TAA) and urinary iodide concentration (UI) was studied in apparently healthy adults in Sapporo (n = 4110) (Sapporo group), and in five coastal areas of Hokkaido (n = 1061) (coastal group) which produce iodine-rich seaweed (kelp). The frequency of above normal UI (high UI) in the morning urinary samples of coastal group was 10.8%, significantly higher than that of Sapporo group (6.4%) (p < 0.001). Frequency of positive TAA in both groups were similar. In Sapporo group TAA was positive in 6.4% of males and 13.8% of females with an age-related increase. The overall prevalence of hyperthyroidism (TSH < 0.15 mU/L) in coastal group (0.6%) was similar to that in Sapporo group (1.1%), while that of hypothyroidism (TSH > 5.0 mU/L) in coastal group (3.8%) was significantly higher than that in Sapporo group (1.3%) (P < 0.001). The frequency of high UI correlated significantly with that of hypothyroidism with negative TAA (r = 0.829, P < 0.05), but not with positive TAA, or with that of hyperthyroidism. Hypothyroidism was more prevalent in TAA negative subjects with high UI than with normal UI. Moreover, serum TSH and thyroglobulin levels were higher and free T4 level was lower in former than in latter group. These results indicate that 1) the prevalence of TAA negative hypothyroidism in iodine sufficient areas may be associated with the amount of iodine ingested, 2) this hypothyroidism is more prevalent and marked in subjects consuming further excess amounts of iodine, and 3) excessive intake of iodine should be considered an etiology of hypothyroidism in addition to chronic thyroiditis in these areas.

Isolation and characterisation of two cDNAs encoding the neuromedin U receptor from goldfish brain.

Intracerebroventricular administration of neuromedin U (NMU) exerts an anorexigenic effect in a goldfish model. However, little is known about the NMU receptor and its signalling system in fish. In the present study, we isolated and cloned two cDNAs encoding different proteins comprising 429 and 388 amino acid residues from the goldfish brain based on the nucleotide sequences of human NMU receptor 1 (NMU-R1) and receptor 2 (NMU-R2). Hydropathy and phylogenetic analyses suggested that these two proteins were orthologues of NMU-R1 and -R2 of goldfish. We established two human embryonic kidney 293 cell lines stably expressing putative NMU-R1 and -R2, respectively, and showed that NMU induced an increase in intracellular calcium concentration ([Ca(2+)](i)) in these cells. We examined the presence of NMU-R1 and -R2 in the goldfish brain by western blotting analysis using affinity-purified antisera raised against peptide fragments derived from these receptors. NMU-R1-specific and NMU-R2-specific antisera detected a 49-kDa and 45-kDa immunopositive bands, respectively, in the brain extract. The mass of each band corresponded to that of the deduced respective primary structures. Reverse transcriptase-polymerase chain reaction analysis showed that NMU-R1 and -R2 transcripts were detected in several tissues. In particular, both mRNAs were strongly expressed in the goldfish brain. By contrast, NMU-R2 mRNA was also expressed in the gut. These results indicate for the first time that NMU-R orthologues exist in goldfish, and suggest physiological roles of NMU and its receptor system in fish.