Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation.

Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7α1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7α1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity.

Is greater than 145{degrees} of deep knee flexion under weight-bearing conditions safe after total knee arthroplasty?: a fluoroscopic analysis of Japanese-style deep knee flexion.

We investigated the characteristics of patients who achieved Japanese-style deep flexion (seiza-sitting) after total knee replacement (TKR) and measured three-dimensional positioning and the contact positions of the femoral and tibial components. Seiza-sitting was achieved after surgery by 23 patients (29 knees) of a series of 463 TKRs in 341 patients. Pre-operatively most of these patients were capable of seiza-sitting, had a lower body mass index and a favourable attitude towards the Japanese lifestyle (27 of 29 knees). According to two-/three-dimensional image registration analysis in the seiza-sitting position, flexion, varus and internal rotation angles of the tibial component relative to the femoral component had means of 148° (SD 8.0), 1.9° (SD 3.2) and 13.4° (SD 5.9), respectively. Femoral surface contact positions tended to be close to the posterior edge of the tibial polyethylene insert, particularly in the lateral compartment, but only 8.3% (two of 24) of knees showed femoral subluxation over the posterior edge. The mean contact positions of the femoral cam on the tibial post were located 7.8 mm (sd 1.5) proximal to the lowest point of the polyethylene surface and 5.5 mm (SD 0.9) medial to the centre of the post, indicating that the post-cam contact position translated medially during seiza-sitting, but not proximally. Collectively, the seiza-sitting position seems safe against component dislocation, but the risks of posterior edge loading and breakage of the tibial polyethylene post remain.

Gefitinib for non-small-cell lung cancer patients with epidermal growth factor receptor gene mutations screened by peptide nucleic acid-locked nucleic acid PCR clamp.

This study was prospectively designed to evaluate a phase II study of gefitinib for non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Clinical samples were tested for EGFR mutations by peptide nucleic acid-locked nucleic acid PCR clamp, and patients having EGFR mutations were given gefitinib 250 mg daily as the second treatment after chemotherapy. Poor PS patients omitted chemotherapy. Of 107 consecutive patients enrolled, samples from 100 patients were informative, and EGFR mutations were observed in 38 patients. Gefitinib was given to 27 patients with EGFR mutations, and the response rate was 78% (one complete response and 20 partial responses; 95% confidence interval: 58-93%). Median time to progression and median survival time (MST) from gefitinib treatment were 9.4 and 15.4 months, respectively. Grade 3 hepatic toxicity and skin toxicity were observed in one patient each. There were significant differences between EGFR mutations and wild-type patients in response rates (78 vs 14%, P = 0.0017), and MST (15.4 vs 11.1 months, P = 0.0135). A Cox proportional hazards model indicated that negative EGFR mutation was a secondary prognostic factor (hazards ratio: 2.259, P = 0.036). This research showed the need for screening for EGFR mutations in NSCLC patients.

Subcellular localization of PP5/TFPI-2 in human placenta: a possible role of PP5/TFPI-2 as an anti-coagulant on the surface of syncytiotrophoblasts.

Placental protein 5 (PP5)/tissue factor pathway inhibitor-2 (TFPI-2), a serine proteinase inhibitor, is homologous to tissue factor pathway inhibitor (TFPI) and commonly found in peripheral blood of pregnant woman. Although TFPI is well known to be synthesized primarily in endothelium and to play an important role in regulation of the extrinsic pathway of blood coagulation, the function of PP5/TFPI-2 remains unclear. Our previous report demonstrated that PP5/TFPI-2 expression is ubiquitous and extremely high in growing placental tissue. Using newly generated polyclonal anti-PP5/TFPI-2 antibody, and by immunohistochemistry and immunoelectromicroscopy, we examined precise localization of PP5/TFPI-2 in placenta especially in syncytiotrophoblasts, which had been shown to produce PP5/TFPI-2 mRNA by our previous study using in situ hybridization. Immunoelectromicroscopy revealed PP5/TFPI-2 localizing on the surface of the microvilli and the membrane of endoplasmic reticulum of syncytiotrophoblasts. To confirm the cell surface association of PP5/TFPI-2, placental villi were incubated with heparin and resultant soluble fraction was analysed by Western blotting. Heparin liberating PP5/TFPI-2 from villi suggested that PP5/TFPI-2 might be retained on the microvilli surface through the binding to membrane-anchored proteoglycans such as glypican and/or syndecan family members. We also examined the relationship between the presence of cell layer of syncytiotrophoblasts and the coagulation using clinical specimens, and revealed that the fibrin depositions were specifically observed on the regions lacking syncytiotrophoblasts cell layer in placental villi. Therefore, it is likely that during pregnancy PP5/TFPI-2 may be retained on the surface of placental villi via proteoglycans, and may play an important role to maintain intervillous blood flow and the patency of microvasculature in feto-maternal blood system mediated by the inhibition of serine proteinases involved in the blood coagulation.

Expression of serine proteinase inhibitor PP5/TFPI-2/MSPI decreases the invasive potential of human choriocarcinoma cells in vitro and in vivo.

OBJECTIVE: PP5/TFPI-2/MSPI is a Kunitz-type serine proteinase inhibitor with broad inhibitory spectra, abundantly produced by placenta and detected in the blood of pregnant women. Expression of PP5/TFPI-2/MSPI is exclusively detected in syncytiotrophoblasts of placenta, but is barely detectable in choriocarcinoma cells, a trophoblast-derived malignant tumor. Chromosome 7, in which the PP5/TFPI-2/MSPI gene is localized, is frequently lost in various types of tumors. We attempted to elucidate the relation between PP5/TFPI-2/MSPI expression and the malignant properties of choriocarcinoma cells. METHODS: Human choriocarcinoma cells, JAR, were transfected with either a human PP5/TFPI-2/MSPI expression vector or an empty vector, and stable clones were obtained. Messenger RNA expression, protein secretion/localization, growth rate, and plating efficiency were evaluated. In vitro migration and invasive activity were determined by transwell chamber experiments. In vivo tumor growth was evaluated by the subcutaneous injection of cells to nude mice and followed by histological examination. RESULTS: Expression of mRNA and protein of PP5/TFPI-2/MSPI were confirmed, and a high producing clone and a low producing clone were chosen for further analysis. The majority of secreted PP5/TFPI-2/MSPI protein was revealed to associate with the extracellular matrix. Expression of PP5/TFPI-2/MSPI did not affect the growth and migration of the tumor cells, but enhanced their plating efficiency. Its expression significantly inhibited invasion through the Matrigel. Invasive growth into the subcutaneous muscle layer was not evident in the nude mouse tumors of the PP5/TFPI-2/MSPI-expressing cells. CONCLUSION: PP5/TFPI-2/MSPI-expressing choriocarcinoma cells showed suppressed potential of invasion in vitro and in vivo. It is suggested that loss or suppression of PP5/TFPI-2/MSPI expression may result in the acquisition of invasiveness in choriocarcinoma cells.

GnRH analog, leuprorelin acetate, promotes regeneration of rat spermatogenesis after severe chemical damage.

BACKGROUND: Future fertility is a major concern for cancer patients who undergo intensive chemotherapy. There has been controversy about whether hormonal treatments may have protective effects against the severe spermatogenic damage caused by chemotherapy or irradiation. Recently, it has been proposed that gonadotrophin-releasing hormone (GnRH) analogs administered after testicular damage stimulate the recovery of spermatogenesis. In this study, we have investigated the effects of GnRH agonist, leuprorelin, on the damage to spermatogenesis induced by busulfan. METHODS: Fisher rats were treated with busulfan, 25 mg/kg, intraperitoneally. The effects of subcutaneous injections of leuprorelin before or after treatment were evaluated histologically 18 weeks later. RESULTS: The percentage of 'recovered' seminiferous tubules was 27.7 +/- 12.6% in control rats without leuprorelin and 26.9 +/- 10.2% in rats with leuprorelin injected 4 weeks before busulfan. Rats in both groups showed poor recovery of spermatogenesis with an increase of intratesticular fluid. However, rats treated with leuprorelin three times (4 weeks apart) after busulfan showed an improvement of up to 56.5 +/- 12.0% (P < 0.05). A focal but massive necrotic lesion in the testis was observed only in this group of rats. CONCLUSIONS: The results demonstrated that leuprorelin administered after chemical testicular damage enhanced the recovery of spermatogenesis. At the same time, a possible significant side-effect of leuprorelin was noted.

Changes of expression level of the differentiation markers in papillary thyroid carcinoma under thyrotropin suppression therapy in vivo immunohistochemical detection of thyroglobulin, thyroid peroxidase, and thyrotropin receptor.

BACKGROUND AND OBJECTIVES: Differences in the expression levels of Thyroglobulin (Tg), Thyroid peroxidase (TPO) and thyrotropin receptor (TSH-R) in primary and recurrent specimens under a suppressive serum TSH condition were elucidated in 26 papillary carcinoma patients. METHODS: Immunohistochemical detection was performed by use of each monoclonal antibody against Tg, TPO, and TSH-R. The staining concentrations of the three markers in each specimen were measured for comparison. RESULTS: The mean staining concentrations of Tg, TPO, and TSH-R in the entire primary tumor were 103.92, 104.6 and 89.25, respectively. Five cases showed stronger expression of all the differentiation markers and eight cases showed weaker expression of all these markers in recurrent tissue than in primary tumors. The weaker expression of TSH-R at the recurrent site as compared with that at the primary site significantly demonstrated the shortness of the disease free interval or overall survival. There were significant differences between the death due to cancer and the weaker expression of TSH-R in the recurrent tumor as compared with that in the primary tumor. CONCLUSIONS: Under the TSH suppressive condition, the markers were not expressed uniformly among recurrent tumors. Even under that state, however, low expression of TSH-R in the recurrent tissue was strongly related to a poorer outcome in the patients.

[A case of advanced breast cancer associated with humoral hypercalcemia that responded to medroxyprogesterone acetate and docetaxel].

A 67-year-old woman with a left advanced breast cancer was admitted to our hospital. Chest CT revealed a parasternal lymph nodal metastasis invading into the sternum, an axillary lymph nodal metastasis, and a lung metastasis. The clinical stage of the patient was i.v. (T4bN2M1). Laboratory examination showed humoral hypercalcemia. After controlling the hypercalcemia with alendronate, sodium hydrate she received chemoendocrine therapy with medroxyprogesterone acetate (MPA) (800 mg/day) and docetaxel (60 mg/body once every three weeks). A complete response was obtained in the primary and metastatic lesions after 3 cycles of docetaxel. This case suggests the efficacy of the combined therapy with MPA and docetaxel on advanced breast cancers.

Semi-quantitative comparison of the differentiation markers and sodium iodide symporter messenger ribonucleic acids in papillary thyroid carcinomas using RT-PCR.

OBJECTIVE: To investigate the levels of expression of the sodium iodide symporter (NIS) and three differentiation markers (thyroglobulin (Tg), thyroid peroxidase (TPO) and thyrotrophin receptor (TSH-R)) in 35 patients with primary (n=31) or recurrent (n=4) papillary thyroid carcinoma, and to compare the findings with clinical data. METHODS: We performed a multiplex semi-quantitative RT-PCR to analyse the relative levels of expression of Tg, TPO and TSH-R mRNAs, and a separate semi-quantitative RT-PCR for NIS mRNA. RESULTS: Tg, TPO and TSH-R mRNAs were expressed in all the patients, whereas NIS mRNA was expressed in all but eight. Analysis of the expression of the differentiation markers in all patients showed a significant correlation among Tg, TPO and NIS. With regard to the relationship between the expression of each gene and the MACIS score, there was significant correlation only for the Tg gene (P<0.05). CONCLUSIONS: The levels of expression of NIS mRNA correlated significantly with those of Tg and TPO mRNAs, but not with those of TSH-R mRNA. The relationship with clinical stage and prognostic score, however, varied among these differentiation markers.

Analyses of the outcome of locally invasive papillary thyroid carcinomas.

We retrospectively analyzed the outcome for patients with locally invasive papillary carcinoma. The study group comprised 40 patients with locally invasive papillary thyroid carcinoma first diagnosed between 1981 and 1995. The enrolled patients were divided into two groups according to whether they underwent complete resection (n = 19) or not (n = 21). All patients were followed-up for a maximum of 206 months and a minimum of 33 months until 1998. There were no significant differences among these two groups with regard to age, sex, or tumor size. Recurrence of the disease was recognized in four patients in the complete resection group and ten in the incomplete group. There were no significant differences in the recurrence rate between both groups. Five patients from the incomplete resection group died of disease, and all patients from the complete resection group were alive. The percentage of surviving patients in the complete resection group was significantly higher than that in the incomplete group. The 15-year survival rates of the complete resection group and incomplete resection group were 100% and 74.2%, respectively. The 15-year survival rate of patients younger than 45 years in the incomplete resection group was 100%. The 15-year survival rate of the complete resection group was significantly higher than that of the incomplete group. In conclusion, complete resection without tumor residue should be performed for patients older than 45 years.

[Treatment and prognosis of T 4 renal cell carcinoma].

BACKGROUND: We studied the cases with T 4 renal cell carcinoma (RCC) to characterize the factors associated with prolonged survival and to clarify the indication of extended nephrectomy. MATERIALS AND METHODS: The study population consisted of 53 patients (44 male and 9 female) with pT 4 RCC treated at the Yokohama City University Hospital and its affiliated hospitals from 1965 to 1994. Survival rates were analyzed with respect to clinicopathological factors (patient age, sex, symptom, tumor growing type, tumor size, histological grade, cell type, structural type, lymph node metastasis, vein invasion, distant metastasis and extended nephrectomy). RESULTS: One-year, 2-years, and 3-years survival rates of the cases with T 4 RCC were 30.4%, 16.4%, and 9.4% respectively. In univariate analysis, improved survival were correlated with no extra-urinary symptom (Logrank: p = 0.0048, Wilcoxon: p = 0.0423), no lymphnode metastasis (Logrank: p = 0.1045, Wilcoxon: p = 0.0199), no distant metastases (Logrank: p = 0.0007, Wilcoxon: p = 0.0006), and enforcement of extended nephrectomy (Logrank: p = 0.0018, Wilcoxon: p = 0.0008). In 28 cases with extended nephrectomy, improved survival was correlated with no extra-urinary symptom, no abdominal wall invasion and no distant metastases. In 5 cases with more than 3 year survival after extended nephrectomy, 4 cases were found to have no distant metastases at the time of operation. Non-operative therapy including interferon for 20 cases without extended nephrectomy were almost ineffective. CONCLUSIONS: These results indicate that if curative excision for T 4 RCC cases without distant metastases could be done, some patients might be appropriate candidates for extended nephrectomy.

[Doxifluridine decreases serum levels of interleukin-6 in a cancer cachexia model].

Cancer cachexia is a common paraneoplastic syndrome in patients with advanced malignancies. However, the mechanisms of the development of cancer cachexia remain to be elucidated. Interleukin (IL)-6 is known to be involved in the development of cancer cachexia. The KPL-4 human breast cancer cell line, which was recently established in our laboratory, secretes IL-6 into the culture medium. Oral administration of doxifluridine (5'-DFUR, 60 mg/kg or 120 mg/kg) significantly inhibited the growth of KPL-4 tumors, reduced the tissue levels of IL-6, and alleviated body weight loss. Serum IL-6 levels were also lowered by 5'-DFUR in nude mice bearing KPL-4 tumors. Additionally, it is suggested that tumor necrosis factor (TNF)-alpha is involved in the cachexia induced by KPL-4 tumors. We suggest that 5'-DFUR suppresses cancer cachexia by lowering IL-6 levels in the tumor tissues and serum.

[Clinicopathological study on patients with renal cell carcinoma according to whole area histological sections].

BACKGROUND: We tried to establish new classification of histological grade and indication of elective nephron-sparing surgery (ENSS) in renal cell carcinoma (RCC), and studied histological changes according to tumor size. METHODS: We made whole area histological sections on 142 cases with RCC and investigated histological aspects and prognosis. RESULTS: (1) To classify the grade of the cases, the worst grade which occupied more than 10% of the tumor provided most appropriate prognosis. (2) In 144 cases (kidneys), satellite tumor lesions (STL) were observed in 69 cases (47.9%), and the cases with vein invasion were observed in 75 cases (51.4%). Incidence of these two factors increased with tumor size. (3) From the point of view of location of STL and vein invasion, ENSS was possible with taking more than 2 cm surgical margin in the cases with less than 4 cm in size and slow growing type. But indication of ENSS should not be decided with ease. (4) Incidence of the cases with multiple structural and cell types and grades increased with tumor size. (5) Incidence of the cases with solid structure, spindle or pleomorphic cell type and high grade increased with tumor size. (6) It was supposed that tumor heterogeneity and progression could be associated with tumor growth in each cases. CONCLUSION: These results suggest the necessity of treatment of the cases with RCC as small as possible. If the tumor is less than 4 cm, prognosis tends to be good, and ENSS might be possible in some cases.

[Prognosis in the cases with renal cell carcinoma according to clinical parameters].

BACKGROUND: The objects of this study is to evaluate the clinical prognostic factors in renal cell carcinoma. MATERIALS AND METHODS: During a 30-year period from January 1965 to December 1994, 1301 cases with renal cell carcinoma were treated at the Yokohama City University Hospital and its affiliated hospitals. In these cases, cause specific 679 cases from January 1965 to December 1990 were analyzed in a study undertaken to investigate long-term treatment results and clinical prognostic factors. RESULTS: 1. The cause specific 5-, 10-, 15-, and 20-year survival rates were 48.7%, 41.1%, 32.3%, and 26.5% respectively, indicating thus that a great number of cases had an ominous prognosis even 5 years or moreafter surgical treatment. 2. Among patients under 40 years of age (n = 29) none died more than 2 years after receiving operation, the prognosis for this particular group of cases being relatively good. 3. Female, incidentally detected cancer, small tumor size (< or = 4.0 cm), slow growing type and low stage were proven to be favourable prognostic factors in renal cell carcinoma. 4. The cause specific 5-year survival rate for the patients (n = 239) from 1965 to 1981 was 33.8%, while the rate for the patients (n = 440) from 1982 to 1990 was 56.5%. This improvement of survival rate was brought by the increase of the incidentally detected renal cell carcinoma. 5. In the incidentally detected renal cell carcinoma, the incidence of slow growing cases and the cases of less than 4.0 cm tumor size were higher than in the symptomatic renal cell carcinoma. 6. Multivariate analysis using Cox's proportional hazard model showed that stage was the most important prognostic factor. CONCLUSIONS: These results suggested that sex, age, symptom, tumor size, growing type, and stage were important prognostic factors in renal cell carcinoma.

Specific expression of PP5/TFPI2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization.

Placental protein 5 (PP5) is a placenta-derived glycoprotein with serine proteinase-inhibiting activity. To date its physiological functions have not been well elucidated. Recently, cDNA sequence analysis revealed that PP5 belongs to the Kunitz-type proteinase inhibitor family and it is identical to tissue factor pathway inhibitor-2 (TFPI-2), homologous to TFPI. Northern blot analysis demonstrated that placental tissue is extremely rich in the transcripts. This study localized PP5/TFPI-2 mRNA in placental tissues at three different gestational periods using in situ hybridization. PP5/TFPI-2 mRNA was specifically detected in syncytiotrophoblast at any gestational period examined, suggesting that syncytiotrophoblast is the principal production site of PP5/TFPI-2 in developing placental tissues. This mRNA expression pattern of PP5/TFPI-2 is quite different from that of TFPI, which is mainly found in vascular endothelial cells. The results indicated possible roles of PP5/TFPI-2 in the trophoblast differentiation and in the maintenance of intervillous blood flow. Also, Northern analysis demonstrated no or little expression of PP5/TFPI-2 in four choriocarcinoma cell lines, in contrast to its abundant expression in syncytiotrophoblast.

Differential expression of trypsin in human ovarian carcinomas and low-malignant-potential tumors.

It is widely recognized that matrix metalloproteinases and serine proteinases play an important role in cancer invasion and metastasis. We have reported that trypsin is synthesized in ovarian carcinomas as well as in some other types of cancers. In general, ovarian cancers easily tend to invade, metastasize, and spread widely into the peritoneal cavity. However, low-malignant-potential (LMP, borderline tumor) ovarian tumors are known to have limited malignant potential for progression, although microinvasion and distant metastasis have been reported among them. To analyze the relationship between varied degrees of trypsin expression and malignant behavior of ovarian tumors, immunohistochemical studies with monoclonal antibodies to human trypsin and clinicopathologic analysis were performed in human ovarian carcinomas, low-malignant-potential tumors, and benign cystadenomas. Thirteen (44.8%) cases of 29 ovarian carcinomas showed prominent trypsin expression, while only 2 (18.2%) cases of 11 LMP ovarian tumors demonstrated low levels of expression. Benign tumors and normal ovaries did not show any positivity for trypsin. These data suggest that tumor-derived heterotropic trypsin may be associated with ovarian tumors in parallel with malignant potential or behavior such as invasiveness or metastasis. At least in some ovarian carcinomas, prominent stromal invasion or metastasis might require the acquisition of or association with tumor-derived trypsin production.

Enhanced expression in seminoma of human zinc finger genes located on chromosome 19.

Six Krüppel-type zinc finger (ZF) genes were cloned from a seminoma cDNA library. One, ZFS-1, showed high sequence homology to the ZNF91 KRAB (Krüppel-associated box) ZF gene family and also the same chromosomal assignment. Interestingly, Northern blot analyses using ZFS-1 and ZNF91 revealed that multiple ZF genes on chromosome 19 were predominantly expressed in seminomas. In addition, the testis and the seminoma showed specific expression of 2.3 kb transcript. Our results suggest that ZF genes on chromosome 19 may be implicated in the development and/or growth of seminomas.

Uterine carcinosarcoma is derived from a single stem cell: an in vitro study.

Expression of intermediate filaments (IFs) has been suggested to be a reliable marker for differentiating epithelial and non-epithelial tumors. Moreover, the c-erbB-2 and p53 genes are considered to be involved relatively early in the process of human carcinogenesis. In order to elucidate the origin of uterine carcinosarcomas, we analyzed IF, c-erbB-2 and p53 expression in and the ultrastructural characteristics of clones derived from a human uterine-carcinosarcoma cell line, EMTOKA. The expression of IFs and other proteins in the EMTOKA clones was identical to that in the EMTOKA cell line. It and its 7 clones all expressed cytokeratins 8, 17, 18 and 19, vimentin, epithelial membrane antigen, S-100, myoglobin, type-II collagen, alpha-smooth-muscle actin, placental alkaline phosphatase and epidermal-growth-factor receptor. The c-erbB-2 and p53 expression levels of all the cell types of the EMTOKA cell line and its clones were the same. Interestingly, an ultrastructural study showed that the EMTOKA cell line and its clones at early and late passages possessed the characteristics of epithelial cell types without either transitional forms between the epithelial and stromal components or differentiation into sarcomatous components. The results of this study lend particular support to the combination tumor hypothesis that a precursor (stem) cell gives rise both to epithelial and to mesenchymal components during the histogenesis of uterine carcinosarcoma, the epithelial component of which appears to be dominant, suggesting that the established cell lines derived from a common stem cell.

Von Hippel-Lindau tumour suppressor gene. Localization of expression by in situ hybridization.

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene is responsible not only for VHL disease, but also for sporadic renal cell carcinoma and cerebellar haemangioblastoma. The distribution of VHL gene expression in the mouse embryo was recently studied by in situ hybridization, along with human VHL in 14-week-old fetal kidney: there was widely distributed expression in the former and expression in the tubules and blastema in the latter. Adult human tissue and other fetal organs were not examined. The present paper describes an in situ hybridization study to assess the function of the VHL gene in adult human tissues and in tissues of human fetus at 28 weeks of gestation. The expression of the VHL gene was limited to the adult and fetal brain and kidney, and the adult prostate. Nerve cells in adult and fetal brain were positive, including the cerebellar Purkinje cells. In adult and fetal kidney, the proximal tubular epithelium, the putative origin of the common type of renal cell carcinoma, showed intense signal, whereas the distal nephron, glomeruli, and nephrogenic blastema showed no significant signal. The prostate showed significant signal in the basal epithelium. The adrenal, pancreas, and epidydimis showed no significant signal, in spite of the frequent occurrence at these sites of neoplastic or hamartomatous lesions in VHL disease.

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos.

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42 degrees-45.5 degrees C and for 10-180 min) was examined. Synthesis of 70 kDa hsp (hsp70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42 degrees C-10 min (236.6 +/- 71.4; P < 0.05) and 43 degrees C-30 min (276.8 +/- 89.4; P < 0.005) compared to control (173.9 +/- 53.9). The 42 degrees C-180 min group (158.0 +/- 27.1 microns) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 +/- 47.3 microns), while heat stress with 43 degrees C for > or = 60 min, 44 degrees-44.5 degrees C for > or = 30 min, or 45 degrees-45.5 degrees C for > or = 10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42 degrees C-180 min, 43 degrees C-10 min, 43 degrees C-30 min, 44 degrees C-10 min, or 45 degrees C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42 degrees C for 180 min, 43 degrees C for 30 min, 44 degrees C for 10 min, and 45 degrees C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear.