A microsatellite genetic linkage map for zebrafish (Danio rerio).

We have constructed a zebrafish genetic linkage map consisting of 705 simple sequence-length polymorphism markers (SSLPs). The map covers 2350 centimorgans (cM) of the zebrafish genome with an average resolution of 3.3 cM. It is a complete map in genetic mapping terms (there is one linkage group for each of the 25 chromosomes), and it has been confirmed by somatic-cell hybrids and centromere-mapping using half-tetrad analysis. The markers are highly polymorphic in the zebrafish strains used for genetic crosses and provide a means to compare genetic segregation of developmental mutations between laboratories. These markers will provide an initial infrastructure for the positional cloning of the nearly 600 zebrafish genes identified as crucial to vertebrate development,and will become the anchor for the physical map of the zebrafish genome.

Proviral insertions in the zebrafish hagoromo gene, encoding an F-box/WD40-repeat protein, cause stripe pattern anomalies.

The zebrafish, Danio rerio, has three types of pigment cells (melanophores, xanthophores and iridophores) and, in adult fish, these cells are organized into a stripe pattern. The mechanisms underlying formation of the stripe pattern are largely unknown. We report here the identification and characterization of a novel dominant zebrafish mutation, hagoromo (hag), which was generated by insertional mutagenesis using a pseudotyped retrovirus. The hag mutation caused disorganized stripe patterns. Two hag mutant alleles were isolated independently and proviruses were located within the fifth intron of a novel gene, which we named hag, encoding an F-box/WD40-repeat protein. The hag gene was mapped to linkage group (LG)13, close to fgf8 and pax2.1. Amino acid sequence similarity, conserved exon-intron boundaries and conserved synteny indicated that zebrafish hag is an ortholog of mouse Dactylin, the gene mutated in the Dactylaplasia (Dac) mouse [1]. The Dac mutation is dominant and causes defects in digit formation in fore- and hindlimbs. This study revealed that the hag locus is important for pattern formation in fish but is involved in distinct morphogenetic events in different vertebrates.

Increased risk of prostate cancer and benign prostatic hyperplasia associated with a CYP17 gene polymorphism with a gene dosage effect.

The CYP17 gene (CYP17) codes for the cytochrome P450c17alpha enzyme, which mediates two key steps in the sex steroid synthesis. There is a polymorphism (a T-to-C substitution) in the 5'-untranslated region, which may influence the transcription level of CYP17 mRNA. There is a continuing controversy as to whether the variant allele is associated with a subset of breast cancer or polycystic ovary syndrome. In prostate cancer research, there are contradictory data concerning the CYP17 risk allele. We explored the association between CYP17 polymorphism and a risk of prostate cancer or benign prostatic hyperplasia (BPH) in a Japanese population. This study included 252 prostate cancer patients, 202 BPH patients, and 131 male controls. A 451-bp fragment encompassing the polymorphic site was amplified by PCR, treated with restriction enzyme MspA1, and electrophoresed on an agarose gel. The MspA1-undigested allele with the published sequence and the MspA1-digested variant allele were designated as A1 and A2, respectively. There was a significant difference (P < 0.05) in the genotypes between prostate cancer patients and male controls, and between BPH patients and male controls. Men with the A1/A1 CYP17 genotype had an increased risk of prostate cancer [odds ratio (OR), 2.57; 95% confidence interval (CI) = 1.39-4.78] and BPH (OR, 2.44; 95% CI = 1.26-4.72) compared with those with the A2/A2 genotype. Men with the A1/A2 genotype had an intermediate increased risk of prostate cancer (OR, 1.45; 95% CI = 0.84-2.54) and BPH (OR, 1.60; 95% CI = 0.89-2.87) compared with those with the A2/A2 genotype. The trend of an increasing risk of prostate cancer and BPH with an increasing number of the A1 allele was statistically significant (prostate cancer versus male control, P = 0.003; OR, 1.57; 95% CI = 1.16-2.12; BPH versus male control, P = 0.008; OR, 1.55; 95% CI = 1.12-2.13). There was no significant association between the CYP17 genotype and the tumor status (grade and stage) of prostate cancer. Our results suggest that the A1 allele of the CYP17 polymorphism is associated with an increased risk of prostate cancer and BPH, with a gene dosage effect. However, the CYP17 genotype does not seem to influence the disease status in prostate cancer.

Association of vitamin D receptor gene polymorphism with prostate cancer and benign prostatic hyperplasia in a Japanese population.

Recent studies have suggested that vitamin D is an important determinant of prostate cancer risk and inherited polymorphisms in the 3'-untranslated region (3'UTR) of the vitamin D receptor (VDR) gene are associated with the risk and progression of prostate cancer. This study was conducted to explore the association of VDR gene polymorphisms with prostate cancer risk in Japanese men who are considered to be much less influenced by environmental risk factors for prostate cancer. We studied 222 prostate cancer patients, 209 benign prostatic hyperplasia (BPH) patients, 128 male controls who were over 60 years old and without any evidence of prostate cancer or BPH, and 198 female controls. A PCR-RFLP method was used to determine three VDR gene polymorphisms in the 3'UTR characterized by restriction enzymes BsmI, ApaI and TaqI. In the BsmI polymorphism, heterozygosity or homozygosity for the absence of the BsmI restriction site was associated with one-third the risk of prostate cancer (P < 0.0001; odds ratio, 3.31; 95% confidence interval, 2.05-5.32) and with one-half the risk of BPH (P < 0.005; odds ratio, 2.07; 95% confidence interval, 1.33-3.22) compared with the male controls. The TaqI and ApaI polymorphisms did not show any significant association with either prostate cancer or BPH. The results indicate that the BsmI polymorphism in the VDR gene plays a significant role in protection against prostate cancer and BPH. Because of the racial difference in the strength of the linkage disequilibrium between the three polymorphisms, additional studies are required to apply the present results to other racial-ethnic groups.

Contact sensitivity to mercuric chloride is associated with I-A region in mice.

Genetic control of contact sensitivity to mercuric chloride antigen was studied using various strains on inbred mice. Mice with class II Ab,d,f,s haplotypes showed a high magnitude of response, whereas those with class II Ak,q were low responders. These results indicate that mercuric chloride contact sensitivity may be influenced by the I-A region of H-2. Transfer experiments revealed that these responses might be mediated by L3T4+, Lyt-2- T cells. However Lyt-2+ T cells do not suppress the DTH response.

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells.

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.

Sepsis increases the plasma membrane content of alpha1 and alpha2 isoforms of Na+-K+ adenosine triphosphatase in rat skeletal muscle.

HYPOTHESIS: Increased Na(+)-K(+) adenosine triphosphatase (ATPase) activity in skeletal muscle during sepsis is caused by transient increases in enzyme content within the plasma membrane. DESIGN: Randomized controlled study. SETTING: University laboratory. INTERVENTION: Eighty-eight adult male Wistar rats were randomly assigned to undergo cecal ligation and puncture (CLP) or sham operation. MAIN OUTCOME MEASURES: Gastrocnemius muscles were harvested 6, 12, 24, and 48 hours after operation and Na(+)-K(+) ATPase activities were measured spectrofluorimetrically. Messenger RNA (mRNA) levels for the alpha1 and alpha2 isoforms of Na(+)-K(+) ATPase were determined by Northern blot analysis. Crude membranes, internal membranes, and purified plasma membranes were isolated from gastrocnemius muscles and protein levels of alpha1 and alpha2 isoforms were determined by Western blot analysis. RESULTS: Na(+)-K(+) ATPase activity in the CLP group was significantly higher compared with the sham group 24 hours after operation (P<.05). However, there were no differences between the sham and CLP groups 6, 12, or 48 hours after operation. No significant differences between the CLP and sham groups were noted in mRNA levels for Na(+)-K(+) ATPase alpha1 and alpha2 isoforms. Western blot analysis revealed that the plasma membrane (but not internal membrane or crude membrane) content of alpha2 and alpha1 isoforms from the CLP group was significantly increased compared with the sham group 24 hours after operation (P<.05). CONCLUSIONS: Na(+)-K(+) ATPase activity increases 24 hours after CLP in gastrocnemius muscle and then declines. This increase is caused by increased Na(+)-K(+) ATPase protein levels in the plasma membrane.

The changes in the activity of pudendal motoneurons in relation to reflex micturition evoked in decerebrate cats.

Reflex micturition was evoked in both non-immobilized (n = 5) and immobilized (n = 5) decerebrate cats by filling the bladder with physiological saline. Intracellular recordings were made from pudendal motoneurons (PU-MNs; n = 14) throughout the periods of before, during and after reflex micturition. The changes in the activity of PU-MNs were correlated with those in the intravesical pressure. Our results support the proposition that coordination of the sphincters and the detrusors is established by a gating mechanism, which is activated by the supraspinal source such as the pontine micturition center.

[Effect of terazosin on lower urinary tract function in the male decerebrate dog].

The effect of terazosin on the lower urinary tract function was studied by combined recording of bladder and urethral pressures and external sphincter electromyogram in 8 male decerebrate dogs. Reflex micturitions were induced by bladder filling before and after terazosin. The statistical analysis was carried out on the urodynamic parameters. During the collecting phase, terazosin at doses of 10, 30 and 100 micrograms/kg produced a significant decrease in maximum urethral pressure in the dose dependent manner. Threshold pressure was significantly shown to decrease at doses of 30 and 100 micrograms/kg. In the urodynamic parameters of the emptying phase there was a significant decrease in maximum contraction pressure at 10 and 30 micrograms/kg, and in voided volume at 100 micrograms/kg. Terazosin seems to facilitate an initiation of the bladder contraction with a decrease in threshold pressure. In concludes that alpha 1 adrenergic activity seems to take an important role for the maintenance of the urethral pressure and to control the initiation of bladder contraction in modulation with threshold pressure.

Effects of growth hormone and insulin-like growth factor-1 on protein metabolism, gut morphology, and cell-mediated immunity in burned rats.

The effects of recombinant human growth hormone (GH) and insulin-like growth factor-1 (IGF-1) were investigated in burned rats. Sprague-Dawley rats were fed exclusively by total parenteral nutrition and were subjected to 20% third-degree scald burns. The rats were then divided into the following three groups: (1) the GH group received GH at a dose of 1 IU.kg-1.d-1 for 2d (n = 10); (2) the IGF group received IGF-1 at a dose of 4 mg.kg-1.d-1 for 2d (n = 19); and (3) the control group received saline (n = 17). Cumulative nitrogen balance increased significantly in the GH (P < 0.01) and IGF (P < 0.01) groups as compared with the control group. There were no differences in nitrogen balance between the GH and IGF groups. Blood glucose was decreased in the IGF group (P < 0.01) and increased in the GH group (P < 0.05) as compared with the control group. The intestinal villus height and wall thickness of the GH and IGF groups were significantly greater than those of the control group. Delayed-type hypersensitivity was enhanced in both the GH and the IGF groups as compared with the control group (both P < 0.01). Furthermore, the increase in the IGF group was significantly greater than that in the GH group (P < 0.05). It was concluded that both GH and IGF-1 improve protein metabolism and immune responsiveness, as well as promote proliferation of the intestinal mucosa.

Oral administration of recombinant human erythropoietin in liposomes in rats: influence of lipid composition and size of liposomes on bioavailability.

Intestinal absorption of recombinant human erythropoietin (Epo) encapsulated in liposomes (Epo/liposomes) was examined by measuring the pharmacological effects of Epo after oral administration in rats. Circulating reticulocyte counts after oral administration of Epo/liposomes showed a profile different from that after intravenous administration. Epo/liposomes 0.1 micron in diameter were absorbed more effectively than those 0.2 micron in diameter. In the 0.1 micron Epo/liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterols (SS), cholesterol (Ch), or soybean-derived sterylglucosides (SG), DPPC/SS (in molar ratio 7/2) and DPPC/Ch (7/2) showed higher efficiency in intestinal absorption than DPPC/Ch (7/4) and DPPC/SG (7/2) at a low dose by the sysmex method. Pharmacological availabilities for oral administration of Epo/liposomes were 0.74-31% and 3.3-30% as evaluated by circulating reticulocyte counts and percentage circulating reticulocytes of erythrocytes, respectively, in comparison to those for intravenous administration of the same dose.

Effect of IGF-1 on protein metabolism in burned rats.

The effects of insulin-like growth factor-1 (IGF-1) on the catabolic and immune response induced by thermal injury were studied in burned rats fed by TPN. An increase of synthesis greater than the increase of breakdown resulted in improved nitrogen retention in the IGF-1 group. There was no effect on the mRNA in the structural proteins of the skeletal muscle and liver. However, the gene expression of albumin and the structural proteins of the diaphragm increased significantly in the rats receiving IGF-1. The proliferation of the gut mucosa and the fractional protein synthesis rate of the small intestine increased, and the endotoxin content of the liver and spleen were smaller in the burned rats that received IGF-1. Delayed type hypersensitivity increased significantly (p < .01) in the IGF-1 group. In conclusion, IGF-1 improved the whole-body protein metabolism, and albumin and respiratory muscle protein synthesis in the burned rats. It significantly promoted the proliferation of the intestinal mucosa, and reduced the intestinal translocation of endotoxin. Cellular immunity was also enhanced.

Distribution characteristics of entrapped recombinant human erythropoietin in liposomes and its intestinal absorption in rats.

Recombinant human erythropoietin (Epo) is frequently administered by intravenous (i.v.) injection for the clinical treatment of renal anemia. Oral (per os; p.o.) administration is desired as an alternative route to i.v. administration, and liposomes have been chosen as a drug carrier. We found previously that after a p.o. administration to rats of Epo entrapped in liposomes before gel filtration, the Epo was absorbed, but variability in the number of days of appearance and in the levels of pharmacological effects, i.e. , the peak of circulating reticulocyte counts (RTC), was observed. The purpose of the present study was to examine the distribution characteristics of Epo in liposomes and intestinal absorption of liposomal Epo in rats by using purified Epo entrapped in liposomes after gel filtration (Epo/liposomes). The distribution characteristics of Epo/liposomes were determined by measuring the Epo in liposomes by a radioimmunoassay, high-performance liquid chromatography and zeta potential measurements. We observed that the protein part of Epo was mostly entrapped in liposomes, and was not adsorbed by the liposomal membrane at middle and high Epo p.o. doses, but the zeta potential of the Epo/liposomes increased negatively with the increase in the Epo p.o. doses. These results suggest that the sialic acid part of Epo entrapped in liposomes may project out from liposomes, depending on the entrapped Epo concentration. Little Epo was adsorbed or penetrated into liposomes when it was added to empty liposomes. After the p. o. administration of Epo/liposomes, the peak of RTC appeared at a 2-day delay on day 6, without variation and without dose dependency in comparison with that after i.v. administration. These results suggest that one of the reasons for the variability may be because the non-entrapped Epo and/or Epo/liposomes itself affected the intestinal absorption of Epo/liposomes. In conclusion, Epo/liposomes without nonentrapped Epo may be clinically useful for the oral administration of Epo.

Effect of tacrolimus and cyclosporine on renal microcirculation and nitric oxide production.

OBJECTIVES: Cyclosporine (CSA) and tacrolimus (TAC) frequently induce nephrotoxicity and similar pathologic changes. Acute CSA-induced nephrotoxicity has been reported to be mediated by activation of vasoconstrictors such as endothelin. The purpose of the present study was to investigate the acute effects of TAC and CSA on the renal microcirculation and upon a vasodilator such as nitric oxide (NO) production. METHODS: Renal blood flow (RBF) in the microcirculation was measured by a Laser Doppler flow meter in uninephrectomised rats. RBF, mean arterial pressure (MAP), and renal vascular resistance (RVR) were measured in the following groups: (a) TAC (0.1 to 2.0 mg/kg/h, n = 3 approximately 6); CSA (20 and 50 mg/kg/h, n = 5); (b) L-NAME (10 mg/kg), an NO synthase inhibitor, 8 minutes prior to TAC (0.5 and 1.5 mg/kg/h, n = 5), or CSA (20 and 50 mg/kg/h, n = 5). Stable NO end-products, serum NO(2) and NO(3), were measured by the Griess method (n = 5). RESULTS: None of the parameters were changed by TAC alone, whereas TAC with L-NAME significantly reduced RBF (-28 +/- 7%) and increased RVR (46 +/- 17%) in a dose-dependent manner. CSA alone significantly reduced RBF (-37 +/- 6%) and increased RVR (69 +/- 22%) without any changes in MAP. The effects of CSA were enhanced by L-NAME. Serum concentration of NO(2) + NO(3) was significantly reduced by both L-NAME alone and CSA (50 mg/kg) (P < .05), while there were no changes with TAC (1.5 mg/kg). CONCLUSIONS: Blockade of NO production enhance the vasoconstrictive effect of CSA, and unmasked such an effect of TAC. These results suggest that the nephrotoxicity of CSA and TAC may involve the NO system.

A case of xeroderma pigmentosum group D determined by photobiological study.

A case of xeroderma pigmentosum group D in 36-year-old woman (XP85TO) is reported. The patient had severe photosensitivity from age 4, and developed multiple basal cell epitheliomas and solar keratoses but exhibited no apparent neurological defects. A skin phototest by monochromatic ultraviolet light revealed a delayed peak of erythema 48 h after irradiation and lowered minimal erythemal doses. Unscheduled DNA synthesis induced in XP85TO cells was 36.0% in dermal fibroblasts and 32.6% in epidermal keratinocytes compared with normal cells. The XP85TO cells were sensitive to ultraviolet killing (n = 1.0, D0 = 0.80 J/m2). In complementation analysis, XP85TO cells did not complement with xeroderma pigmentosum group D cells. These results indicate that patient XP85TO had xeroderma pigmentosum group D. The Japanese group D patients including XP85TO case showed delayed onset of skin malignant tumors and neurological abnormalities, compared with the group D patients in Europe and the United States. These findings suggest a possible ethnic variation of the clinical phenotype, despite the similar repair defect and ultraviolet hyperssensitivity.

Cutaneous leiomyosarcoma.

A child's head-sized tumor on the upper back developed in a 43-year-old man. In spite of an extensive operation for the tumor, he died of multiple metastasis 15 months after the operation. Histologically, tumor cells proliferated throughout the dermal and subcutaneous regions, and a boxed-in appearance was noted with silver staining. Because electron microscopic observations strongly suggested a smooth muscle origin, we diagnosed this case as cutaneous and subcutaneous leiomyosarcoma.

Rectosigmoidal bladder utilizing an intussuscepted ileal segment: a surgical technique for urinary diversion.

A novel technique of urinary diversion was designed by interposition of an intussuscepted ileal segment between the ureters and the rectosigmoidal pouch, thus preventing ureteral reflux as well as stenosis at the uretero-enteric anastomosis, possible occurrence of urocolonic carcinoma, and frequent evacuation or incontinence. Since all the procedures were limited to the lower abdominal cavity, the surgical invasiveness was compatible with that of an ileal conduit. Our early experience in 15 patients showed that this technique can be considered for those in whom the urethra is not available.

Total cystoprostatectomy in the treatment of locally advanced prostate carcinoma.

OBJECTIVE: Locally advanced prostate carcinoma frequently causes lower urinary tract symptoms and is a clinical challenge when radiation and/or hormone therapy fail. We investigated whether cystoprostatectomy with urinary diversion benefits patients with locally advanced prostate carcinoma in terms of quality of life and prognostic outcome. PATIENTS AND METHODS: Between 1989 and 2001, we performed 15 cystoprostatectomies for stage C-D1 prostate carcinoma with bladder neck involvement. Of these patients, 5 underwent ileal conduit, 8 rectal bladder, 1 Koch pouch, and 1 ureterocutaneostomy. All the patients received neoadjuvant and/or adjuvant hormonal therapy. In the same period, 28 patients underwent retropubic prostatectomies and 15 patients received hormone therapy alone for stage C-Dl disease. These patients were included as references. RESULTS: Lower urinary tract symptoms caused by bladder involvement were controlled well until the end of follow-up for all the patients in the cystoprostatectomy group. There was no statistically significant difference in QOL score assessed with the EORTC QLQ-C30 questionnaire between the prostatectomy group and the cystoprostatectomy group, while that in the hormone therapy group was lower than those in the surgery groups. There was no statistically significant difference in 5-year PSA-relapse-free survival among cystoprostatectomy, prostatectomy, and hormone therapy groups. Patients in the hormone therapy group died earlier than those in the prostatectomy group (p = 0.02), while those in the cystoprostatectomy group did not. CONCLUSION: These results suggest that total cystoprostatectomy with urinary diversion is a valid option, in terms of disease control and QOL, for prostate cancer patients whose tumor is infiltrating into the bladder.

[Identification of effective region of the pons in response to inaperisone which facilitates urine storage].

To identify the effective region of the pons in response to inaperisone which facilitates urine storage, inaperisone (100 mM, 0.2 microliters) was injected into the nucleus locus coeruleus alpha (LCa, the pontine micturition center), the nucleus locus subcoeruleus (LSC, the pontine urine storage center) and the nucleus reticularis pontis oralis (PoO, micturition inhibitory region) of the decerebrate cats. On reflex micturition, inaperisone injection into the LSC decreased voiding volume, and increased residual volume and bladder capacity, significantly. However, there was no difference in the maximum bladder pressure before and after inaperisone injection into the LSC. Inaperisone injection into the LCa or the PoO had no influence on reflex micturition. These results suggest that effective region of the pons in response to inaperisone is the LSC, and that inaperisone facilitates the urine storage neural mechanism in the LSC.