Biological characteristics of fish germ cells and their application to developmental biotechnology.

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Differential localization of protein kinase C isozymes in retinal neurons.

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.

A newly synthesized bifunctional inhibitor, W-77, enhances adriamycin activity against human ovarian carcinoma cells.

A newly synthesized calmodulin antagonist, (S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxy-carbonylpiperazinyl++ +)-1-(P-methoxybenzyl)ethyl]-N-methylbenzenesulfonamide dihydrochloride (W-77), acts as a calcium-independent uncompetitive antagonist which binds to glutathione-S-transferase (GST). We purified GST from human placenta using drug affinity chromatography on a column of W-77 coupled with Sepharose 6B and demonstrated that W-77 bound to GST. A spectrophotometric assay also showed that W-77 inhibited GST activity. We prepared Adriamycin-resistant and -sensitive cells from human ovarian serous cystadenocarcinomas. Immunoblot analysis revealed that GST expression was increased in the Adriamycin-resistant cells. We also purified GST from Adriamycin-resistant cells and found that W-77 bound to the GST obtained from these ovarian carcinoma cells. Adriamycin resistance was partially overcome by the addition of W-77 (10 microM) to the cultured cells. In addition, we investigated the effect of W-77 on P-glycoprotein. Northern blot analysis revealed MDR1 gene expression in Adriamycin-resistant cells. Although W-77 was less potent in increasing the intracellular Adriamycin content than verapamil, it was more effective in overcoming Adriamycin resistance. These results suggest that W-77 enhances the antitumor activity of Adriamycin by inhibiting both GST and P-glycoprotein.

Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase.

Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.

Phosphorylation of caldesmon.

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulin-dependent protein kinase II.

Distinct regional localization of neurocalcin, a Ca(2+)-binding protein, in the bovine adrenal gland.

Neurocalcin (molecular weight 23,000 and 24,000) is a Ca(2+)-binding protein with three putative Ca(2+)-binding domains and is present in large amounts in nervous tissues. Neurocalcin isoproteins separated by C18 reverse-phase column chromatography are insoluble in buffer solution and it is impossible to determine the dissociation constant of neurocalcin with Ca2+. To overcome this difficulty, recombinant neurocalcin was synthesized, based on one of the cDNAs of the neurocalcin isoproteins. Stoichiometric titration experiments, using recombinant neurocalcin, indicated that this protein bound 2 mol Ca2+/mol protein and that the apparent dissociation constant for Ca2+ was 2.2 mumol/l, suggesting that neurocalcin plays a physiological role in cellular function. Immunoblotting showed that neurocalcin is present in the bovine adrenal gland in addition to the nervous tissues. Neurocalcin, identified by immunoblotting, was purified from the bovine adrenal gland. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of neurocalcin from the bovine brain showed 23 kDa and 24 kDa double bands, while SDS-PAGE of neurocalcin from the adrenal gland showed a single band of apparently 24 kDa, suggesting that the expression of neurocalcin isoproteins differs from tissue to tissue. The content of neurocalcin in the adrenal gland was 10 micrograms protein/100 g wet tissue. Immunohistochemical analysis showed the occurrence of neurocalcin in zona glomerulosa and adrenal medulla but not in zona fasciculata or zona reticularis. The restricted localization of neurocalcin in the adrenal gland suggests that a similar Ca2+signal pathway may be present in zona glomerulosa a nd the adrenal medulla.

Immunochemical studies of the turnover of delta-aminolevulinate synthase in rat liver mitochondria and the effect of hemin on it.

The half-life of delta-aminolevulinate (ALA) synthase in the liver mitochondria of allylisopropylacetamide-treated rats was estimated to be about 35 min, taking advantage of the observation that hemin strongly inhibits the translocation of ALA synthase from the liver cytosol into the mitochondria. ALA synthase accumulating in the liver cytosol in allylisopropylacetamide-treated rats is rapidly translocated into mitochondria with a half-disappearance time of about 20 min, but does not appear to undergo degradation until it is translocated into mitochondria. A study with an ALA synthase-specific IgG revealed that the decrease in the ALA synthase activity in the liver mitochondria observed after the administration of cycloheximide or hemin effectively paralleled the decrease in the amount of immunologically reactive protein, providing further evidence that ALA synthase is rapidly degraded in the mitochondria.

Effects of hemin on the synthesis and intracellular translocation of delta-aminolevulinate synthase in the liver of rats treated with 3,5-dicarbethoxy-1,4-dihydrocollidine.

The administration of hemin to rats which had been treated with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) brought about a rapid decrease of the delta-aminolevulinate (ALA) synthase activity in the mitochondria and an appreciable increase of the enzyme activity in the cytosol in the liver. In DDC-treated rats, however, the degree of accumulation of ALA synthase in the liver cytosol resulting from the hemin administration was relatively small and the total ALA synthase activity in the liver was considerably decreased after the hemin administration; these results are in contrast with those for allylisopropylacetamide-treated rats. Further studies by combined use of [3H]leucine and an anti-ALA synthase IgG revealed that incorporation of [3H]leucine into the mitochondrial ALA synthase was greatly reduced in hemin-injected rats and that incorporation of [3H]leucine into total ALA synthase in the liver (the sum of radioactivity incorporated into the mitochondrial enzyme and the cytosolic enzyme) was also decreased to a considerable extent in these rats; this decrease in incorporation of [3H]leucine appeared to occur immediately after the administration of hemin. It was concluded that hemin actually inhibits the translocation of ALA synthase from the cytosol into the mitochondria and that hemin also inhibits de novo synthesis of ALA synthase, possibly at a post-transcriptional level in DDC-treated rats.

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.

Relationship between Joint National Committee-VI classification of hypertension and ambulatory blood pressure in patients with hypertension diagnosed by casual blood pressure.

BACKGROUND: White-coat hypertension has been diagnosed arbitrarily based on different criteria. In 1997, the Joint National Committee-VI (JNC-VI) reported a new classification of hypertension and strongly emphasized the importance of ambulatory blood pressure (ABP) monitoring. The report pronounced normal ABP values for the first time. HYPOTHESIS: The study's aim was to clarify the relationship between casual blood pressure (BP) and ABP of patients with essential hypertension in each stage of JNC-VI classification, and the prevalence of white-coat hypertension diagnosed by using JNC-VI normal ABP criteria. METHODS: Ambulatory blood pressure was monitored non-invasively in 232 patients with essential hypertension whose casual BP was > or = 140/90 mmHg. The patients were classified according to JNC-VI classification, and their casual BP was compared with ABP. The criterion of white-coat hypertension was defined as casual BP > or = 140/90 mmHg with normal ABP according to JNC-VI criteria (< 135/85 during daytime and < 120/75 during nighttime). RESULTS: Mean ABP increased as the stage advanced, and the differences between casual BP and ABP also increased. There were considerable overlaps in the distribution of ABP among stages. The prevalence of white-coat hypertension was 13% overall: 30% of the patients with isolated systolic hypertension, 19% of those in stage 1, 10% in stage 2, and 4% in stage 3. CONCLUSIONS: Classification of hypertension based on casual BP may not always correspond in severity to that based on ABP. Ambulatory blood pressure monitoring recommended by JNC-VI is very useful for the evaluation of hypertension to differentiate white-coat hypertension from true hypertension.

Hypergravity modulates behavioral nociceptive responses in rats.

Hypergravity (2G) exposure elevated the nociceptive threshold (pain suppression) concomitantly with evoked neuronal activity in the hypothalamus. Young Wistar male rats were exposed to 2G by centrifugal rotation for 10 min. Before and after 2G exposure, the nociceptive threshold was measured as the withdrawal reflex by using the von Frey type needle at a total of 8 sites of each rat (nose, four quarters, upper and lower back, tail), and then rats were sacrificed. Fos expression was examined immunohistochemically in the hypothalamic slices of the 2G-treated rats. When rats were exposed to 2G hypergravity, the nociceptive threshold was significantly elevated to approximately 150 to 250% of the 1G baseline control levels in all the examination sites. The 2G hypergravity remarkably induced Fos expression in the paraventricular and arcuate nuclei of the hypothalamus. The analgesic effects of 2G hypergravity were attenuated by naloxone pretreatment. Data indicate that hypergravity induces analgesic effects in rats, mediated through hypothalamic neuronal activity in the endogenous opioid system and hypothalamo-pituitary-adrenal axis.

[Synthesis, pharmacological activity and biopharmaceutical characteristics of alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetates].

The pro-drugs of alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetic acid(I) with a potent anti-inflammatory activity were synthesized in order to reduce its gastrointestinal side effects. Various esters synthesized were evaluated for their anti-inflammatory activity and ulcerogenicity. Among the compounds maintaining a potent activity of I, N,N-dimethylcarbamoylmethyl alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetate (II-18) showed excellent biopharmaceutical characteristics. The ulcerogenic effect of II-18 on the rat gastric mucosa was about 3 times less than that of I. It was suggested that II-18 may be an useful biolabile pro-drug for I among the compounds tested.

[Pulmonary infarction diagnosed by transbronchial lung biopsy].

A 43-year-old man complained of chest pain, fever, and hemosputum in February 1997. Chest X-ray films revealed small opacity in the right lower lung field. The patient received therapy for pneumonia and his condition gradually improved. However, on March 21 he was admitted to our hospital with worsened chest pain and radiographic findings. A transbronchial lung biopsy revealed thrombus and necrosis, thus yielding a diagnosis of pulmonary infarction. The patient did not exhibit any underlying disease or coagulation abnormalities. Treatment with ticlopidine resulted in a favorable course. This was a rare case of pulmonary infarction in which TBLB findings led to the diagnosis.

[Advance in eosinophil chemotaxis assay method--automatic count of migrated cells by the image analyzing system in the Boyden method].

Eosinophils, which are prominent inflammatory cells in the asthmatic airway, are attracted to the airway by several chemoattractants. At present, eosinophil chemotaxis is studied employing the Boyden Millipore chamber system in vitro. In the Boyden method, the number of migrated cells are evaluated by direct microscopic observation of individual cells. This microscopic procedure usually requires much time and it is difficult to obtain objective results. Therefore, we have developed a new fractional measurement technique, using the image analyzing system connected with a color video camera and a computer for counting the eosinophils which have migrated into a filter. Using this system with the Boyden method, migrated cells were observed more objectively and quickly. Therefore, this image analyzing system with the Boyden method, may be a useful technique for the fractional measurement of migrated eosinophils in a sample mixture of eosinophils and neutrophils.