Regulation of prostate cell growth and morphogenesis by Dickkopf-3.

Wnt signalling plays a critical role in the development of cancer. Recent studies indicate that Wnt signalling is negatively regulated by secreted Wnt antagonists such as secreted frizzled related proteins (sFRPs) and Dickkopfs (Dkks). We compared Dkk family expression levels in normal prostate and prostate cancer cells and found a reduction in Dkk-3 expression in cancer cells. Ectopic expression of Dkk-3 inhibited colony formation in LNCaP and PC3 prostate cancer cell lines and inducible expression of Dkk-3 reduced LNCaP cell proliferation. Moreover, small interfering RNA-mediated downregulation of Dkk-3 enhanced cell cycle progression in untransformed RWPE-1 prostate epithelial cells. Immunohistochemical analysis revealed that Dkk-3 is expressed in a subset of normal prostate gland acini and that Dkk-3 expression is reduced in prostate tumours, particularly those with a high Gleason grade, suggesting a role for Dkk-3 in postmitotic differentiation. Consistent with this, depletion of Dkk-3 disrupted acinar morphogenesis of RWPE-1 cells in a three-dimensional cell culture model. Our results are consistent with the loss of Dkk-3 expression resulting in impairment of glandular structure and uncontrolled prostate epithelial cell (PrEC) proliferation, both of which are crucial for prostate cancer progression.

Differences in bone and vitamin D metabolism between primary hyperparathyroidism and malignancy-associated hypercalcemia.

Bone and vitamin D metabolism are examined in patients with primary hyperparathyroidism (1 degree HPT), humoral hypercalcemia of malignancy (HHM), and local osteolytic hypercalcemia (LOH) with normal renal function. Among the bone resorption markers, T scores of total deoxypyridinoline (Dpyd) were highest in HHM and were significantly higher than those in 1 degree HPT. Among the formation markers, T scores of osteocalcin (OC) were highest in 1 degree HPT but were negative in HHM. The elevation in total Dpyd was associated with an increase in OC in 1 degree HPT, and the ratios of total Dpyd/OC were similar to those in controls. In contrast, many patients with HHM and LOH exhibited elevated total Dpyd and suppressed OC with increased total Dpyd/OC ratios, but the ratios varied widely. Serum 1,25-dihydroxyvitamin D [1,25(OH)2D] was elevated in 1 degrees HPT but was suppressed in HHM and LOH at any serum Ca levels. These results demonstrate that increased bone resorption is associated with enhanced bone formation in 1 degrees HPT but are uncoupled in many of the HHM and LOH patients, and that total Dpyd/OC ratio can be a useful index to estimate the coupling state of bone. It is suggested that the reduction in serum 1,25(OH)2D cannot be explained by an elevation in serum Ca in HHM and LOH, and that the differences in bone and vitamin D metabolism in HHM and LOH from those in 1 degree HPT may be caused by a common mechanism such as the secretion of some cytokines from tumors.

Immunohistochemical localization of procollagen types I and III during placentation in pregnant rats by type-specific procollagen antibodies.

To examine the sequential localizations of procollagen Types I (Pro I) and III (Pro III) during chorioallantoic placental formation in pregnant rats, we prepared polyclonal anti-rat Pro I- and III-specific antibodies. Biochemical analysis of a fraction containing [14C]-glycine-incorporated collagen from pregnant rat uteri showed that collagen Types I and III were actively synthesized during placental development. We examined 8-, 9.5-, 13-, and 20-day gestation rat uteri immunohistochemically. At Days 8 and 9.5, in the basal decidua facing the fetal cytotrophoblastic giant cell layer and implantation site, the immunoreactivity for Pro I was higher than that for Pro III. On Day 13, the enlarged myometrium and cytotrophoblastic cell layer showed increased immunoreactivity for Pro III. Unexpectedly, polygonal trophoblastic cells invading and modifying the maternal central artery showed intense immunoreactivity for Pro III. On Day 20, the fetal mesenchyme, large fetal blood vessels, and subendothelial stroma, including fetal blood capillaries, were more immunoreactive to Pro III antibody than to Pro I antibody in the labyrinth. Pro I and III synthesis and processing appear to be developmentally regulated and may be related to control of the microenvironment for supporting the fetus, control of the maternal blood supply stabilizing the fetoplacental physiological functions, and parturition.

Rapid expression of collagen type X gene of non-hypertrophic chondrocytes in the grafted chick periosteum demonstrated by in situ hybridization.

We investigated the spatiotemporal localization of collagen Type I, II, and X mRNAs in the subcutaneously grafted chick periosteum by in situ hybridization. Five days after transplantation, we noted three types of histological findings in the grated tissue. (a) Developing trabecular bone exhibited proliferation of spindle-shaped fibroblastic cells and polygonal osteoblasts with moderate signals for collagen Type I mRNA. (b) Developing cartilage contained ovoid chondrocytes with a moderate level of both collagen Type I and II mRNAs. Differentiating chondrocytes with increased collagen Type X mRNA developed during the course of endochondral ossification. (c) An atypical mass of cartilage weakly stained with alcian blue was composed of a large number of non-hypertrophic chondrocytes exhibiting high signals for collagen Type X mRNA. At Day 9, we observed the typical histological features of both membranous and endochondral ossification. However, sparsely distributed chondrocytes with high signals for collagen Type X mRNA were also demonstrated in osteoid and/or woven bone. The phenotype of chondrocytes showing rapid expression of collagen Type X gene derived from grafted periosteum seems to participate in the important role of endochondral bone formation in the early stage of fracture repair.

Homocysteine as a risk factor for restenosis after coronary angioplasty.

We examined the relationship between plasma homocysteine levels and restenosis after PTCA (Percutaneous transluminal coronary angioplasty) to investigate whether plasma homocysteine levels can be a predictor of restenosis after PTCA. One hundred and twelve male patients who have undergone a successful elective PTCA were consecutively enrolled and plasma homocysteine levels were measured at the time of follow-up angiography. Plasma homocysteine levels in patients with restenosis were significantly higher than those in patients without restenosis (15.0 +/- 3.9 vs. 13 +/- 2.9 micromol/L; P = 0.011). The difference was augmented when diabetic patients were selectively studied. The comparison between restenosis group and non-restenosis group indicated the threshold effect of hyperhomocysteinemia. These results suggest that plasma homocysteine is a potential risk factor of restenosis after PTCA, and therapeutic strategy targeted against hyperhomocysteinemia may be beneficial for preventing restenosis.

Characterization of high molecular weights of complexes and polymers of cytoplasmic proteins in Escherichia coli.

To search for filamentous polymers of cytoplasmic proteins of Escherichia coli, high molecular weights (> 670 kDa) of protein complexes of cell extracts were fractionated by gel filtration and ion-exchange column chromatography. Proteins of 100, 77 and 52 kDa were co-purified. The 100- and 52-kDa proteins were identified to be pyruvate dehydrogenase and lipoamide dehydrogenase, respectively, by determining the N-terminal amino acid sequences. Experimental results indicate that the 77-kDa protein is identical to dihydrolipoamide acyltransferase. The 100-kDa protein was found to be identical to the 100-kDa protein described by Tomioka (1991), and was related to the formation of filaments and sheets in the presence of 100 mM KCl. However, neither long filaments nor sheets were observed in our sample containing these enzymes, which was not consistent with Tomioka's conclusion. Another 100-kDa protein which forms spirosome-like particles was purified and identified to be alcohol dehydrogenase based on the N-terminal sequence.

Synthetic reaction of Cellvibrio gilvus cellobiose phosphorylase.

The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.

Medical and surgical aspects of parathyroidectomy.

A more logical approach to the management of the chronic dialysis patient with parathyroid hyperplasia has become possible thanks to recent progress in cellular and molecular analysis of surgically removed parathyroid glands and accumulation of clinical experience. When one or more parathyroid glands progress to the stage of nodular hyperplasia, it is usually difficult to control PTH secretion even by calcitriol pulse therapy. For such patients, we have developed two new therapeutic approaches, i.e., selective percutaneous ethanol injection therapy (PEIT) and direct calcitriol injection therapy, in combination with medical therapy. For optimal selection of therapeutic modalities it is indispensable to evaluate the degree and stage of parathyroid hyperplasia. For successful management, prevention of nodular hyperplasia is the most important strategy.

Transmission of Rickettsia orientalis to man by Leptotrombidium akamushi at a scrub typhus endemic area in Akita Prefecture, Japan.

During the summer seasons of 1956 through 1970, 93 larval trombiculid mites were removed from 386 individuals who had been bitten by chiggers in Jumonji, Akita Prefecture. All 87 larvae that were available for examination were identified as Leptotrombidium akamushi. Infestation of man occurred predominantly during July and August, but the period extended from June to November. The duration of attachment was approximately 1 to 3 days. Usually only 1 chigger was found on a victim but on occasion as many as 7 were removed at one time. Scrub typhus developed in 45 (11.7%) of the 386 farmers bitten by chiggers. Although patients with scrub typhus may have sustained multiple bites, only one eschar was found. However, 76% of the patients did not recognize a bite either at the site where an eschar subsequently appeared or elsewhere prior to the onset of disease. If it is assumed that workers who did not subsequently become ill were equally unaware of chigger bites, then the probable incidence of disease in chigger victims was 3.1%. The minimum infectivity rate of L. akamushi larvae in Akita Prefecture was estimated to be 2.3%. Based upon the assumption that infection was transmitted as the result of a single bite, the infective rate of chiggers attacking man was calculated to be 2.5%.

Technical aspects and outcome of in situ right internal thoracic artery grafting to the major branches of the circumflex artery via the transverse sinus.

BACKGROUND: Little is known about the anatomic limitations of in situ right internal thoracic artery (RITA) grafting to the circumflex artery. METHODS: To evaluate the technical aspects and outcome of revascularization of the proximal and distal major branches of the circumflex artery (obtuse marginal [OM] branch and posterolateral [PL] branch), a total of 145 patients who possessed a graftable branch of the circumflex artery were enrolled into the prospective project. There were 73 patients who had the PL branch as a primary target and 72 patients with OM branches, which were allocated by a blinded observer who reviewed the preoperative angiography. RESULTS: Changes of primary target vessels were required in 9 patients (6.2%), yielding an overall success rate of RITA grafting of 93.8%. The success rates of RITA grafting to the OM branch and the PL branch were 95.8% (69/72; CI 88.3% to 99.1%) and 91.7% (67/73; CI 83.0% to 96.9%), respectively. The univariate analysis identified grafting under hypothermic ventricular fibrillation as predictors of inability to use in situ RITA grafting for revascularization of the circumflex artery. RITA grafting to the PL branch is not identified as a predictor. Postoperative angiography in 136 patients revealed only one occlusion (0.75%) of the RITA graft anastomosed to the marginal artery. There were no significant differences in patency rates between left and right ITA grafts. CONCLUSIONS: This prospective study showed that in situ RITA was, in most cases, able to reach most branches of the major circumflex artery and demonstrated an excellent patency rate.

The impact of chronic renal failure on atherosclerosis of the internal thoracic arteries.

BACKGROUND: Little is known about the impact of renal failure on atherosclerotic changes in the internal thoracic artery (ITA). METHODS: A total of 20 consecutive patients on chronic dialysis who underwent coronary artery bypass grafting (CABG) during April 1998 through September 1999 were investigated. The 20 control patients were selected from the same interval to rigorously match risk factors. Atherosclerosis of the ITA collected from each patient was analyzed using the subjective evaluation proposed by Kay and colleagues. RESULTS: There were no cases of greater than 25% atherosclerotic luminal narrowing among a total of 35 ITA specimens from dialysis patients. The degree of atherosclerosis was not significantly different from that of the specimens from matched patients (p = 0.18). No calcification was found in ITA grafts either microscopically or macroscopically. The number of elastic lamellae, an index of the elasticity of the ITA graft, was not significantly different from those obtained from the matched patients. Analysis of preoperative coronary angiography revealed that coronary calcification was significantly more frequent in dialysis patients (15 patients, 75%) than in matched patients (p < 0.05). By analysis of postoperative angiography in dialysis patients, no evidence of atherosclerotic changes was found in 28 opacified ITAs. In addition, despite the presence of calcification in the native coronary, no calcification was evident along the entire length of the ITAs. CONCLUSIONS: This study revealed the minimal impact of chronic renal failure on atherosclerotic changes in the ITA. The results of this study support the continued use of ITA grafting in dialysis patients.

Metastases from carcinoma of the colon and rectum detected in small lymph nodes by the clearing method.

A study of regional lymph node metastases was performed using the clearing method in 322 patients with carcinoma of the colon and rectum (140 with carcinoma of the colon and 182 with carcinoma of the rectum) who had undergone surgical resection. The mean number of nodes examined per patient was 76.4 and the metastatic rate (patients with metastases divided by the total of patients) was 61.4 percent, with a metastatic incidence (nodes with metastases divided by the total of examined nodes) of 6.4 percent for carcinoma of the colon using the clearing method. For carcinoma of the rectum, the mean number of nodes examined was 73.7 with a metastatic rate of 57.1 percent and a metastatic incidence of 7.1 percent. In contrast, node analysis by the conventional manual method resulted in a mean of 18.1 nodes being examined, with a metastatic rate and incidence of 42.1 and 12.8 percent, respectively, for carcinoma of the colon. Manual examination of lymph nodes in carcinoma of the rectum resulted in a mean of 21.2 nodes being examined, with a metastatic rate and incidence of 50.0 and 16.8 percent, respectively. Compared with the manual method, the clearing method provided a greater number of nodes, a higher metastatic rate and a lower metastatic incidence. These differences may be explained by the detection of metastatic regional nodes smaller than 4 millimeters in maximum diameter by the clearing method. By TNM classification there were more pN3 than pN2 lesions. The five year survival rate after curative resection was 78.5 percent for pN1 lesions, 45.7 percent for pN2 lesions and 45.4 percent for pN3 lesions for carcinoma of the colon and 72.7 percent for pN1 lesions, 75.0 percent for pN2 and 53.9 percent for pN3 lesions for carcinoma of the rectum. There was no significant survival difference between the patients with pN1, pN2 and pN3 carcinomas. The presence of regional nodes metastases should be examined in detail. Therapies and prognosis of carcinoma of the colon and rectum should be discussed based on accurate staging.

Immunohistochemical studies of basement-membrane in colorectal-cancer and primary cultured-cells.

The basement membrane (BM), a structure of crucial importance in the invasion and metastasis of cancers, was studied immunohistochemically using colorectal cancer tissues. In addition, BM-like structures formed in the primary culture of cancer cells were compared to the BM of primary cancers. BM was stained with antibodies specific for laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan. In the normal colon these compounds were detected in the glandular BM, vascular BM and perinervium by immunostaining. Components of the BM were not stained in some cancer tissues, and BM staining essentially disappeared in the invasive front of cancer in several cases. Of whole cancer tissues, 10 of 23 cases (43.5%) were BM positive and the remaining 13 cases (56.5%) were negative. A three dimensional primary culture of colorectal cancer cells was performed in collagen gel. The cancer cells obtained from primary cancers which had densely stained for BM formed a BM-like structure in primary culture, but those from negatively stained cancer tissues showed no BM-like structures. The BM-like structure in the primary culture was shown to have the same components as the true BM by immunohistochemistry, and this membranous structure was shown to have originated in the cancer cells. In the primary culture without any BM-like structures, granules containing BM components were observed around tumor cell clusters. The primary culture of tumor cells in collagen gel is closely related to the original tumor tissues and therefore may provide a good model system for the investigation of BM in cancer.

Lipaemia retinalis in a 29-day-old infant with type 1 hyperlipoproteinaemia.

A 29-day-old infant born of consanguineous parents had type 1 hyperlipoproteinaemia associated with lipaemia retinalis. Biochemical analyses disclosed hypertriglyceridaemia, hyperchylomicronaemia, and lipoprotein lipase deficiency. Ophthalmoscopically all retinal vessels had the creamy appearance of lipaemia retinalis. The plasma lipoprotein levels and fundus condition became normal after her ingestion of fat was limited. To our knowledge this is the youngest patient reported with type 1 hyperlipoproteinaemia associated with lipaemia retinalis.

Multi-beam polarized photometric detector for high-performance liquid chromatography.

A novel multi-beam polarized photometric detector (PPD) for high-performance liquid chromatography (HPLC) is described. By pairing a polarizing prism with a thin quartz plate as a retarder, many linear polarized beams are produced at every 1/2 wavelength of the plate, and the polarizing axes of the adjacent beams intersect each other. The addition of another prism inclining its polarizing axis by pi/4 against the first one enables the simultaneous measurement of optical rotations based on the PPD at many wavelengths. The combination of these optics with a photo-diode array detector can be used to construct a modulated type polarimeter. This detector is designed to measure the optical rotation of an analyte at its absorption band. The spline function connecting the points at 1/4 wavelengths of the plate was used as a baseline to extract the PPD waves. The use of the similarity factor as a noise filter gave high sensitivity. Application of the proposed technique to an analyte carrying the Cotton absorption band provided good results.

Endosteal new bone formation in the long bones of adjuvant treated rats.

Histopathological changes were examined mainly in the diaphyseal parts of long bones, especially femur in adjuvant-treated male Lewis-SPF rats, with reference to clinical symptoms of chronic osteoarthritis. The diaphyseal bone marrow of long bones in these rats sequentially showed three different processes of chronic pathological changes, which, however, partly overlapped each other. Initially, multiple epitheloid cell granulomas or granulomatous lesions containing fibrin deposits began to appear in the marrow space about 22 days after adjuvant injection, when the joint score of arthritis reached a peak in severity. Secondly, about a week after appearance of the granulomas, there occurred the intramembranous endosteal new bone formation proceeding from the endosteum towards the granulomatous lesions. The bone formation reached a maximum about 64 days after the treatment, when the redness of joints of feet and hands was already sedated. Finally, about 40 days after occurrence of the second event, the newly growing bone matrix began to be actively resorbed simultaneously. On the other hand, in the bone marrow of metaphyseal parts of long bones in these rats, severe acute osteomyelitis was observed from an early stage, with marked destruction of bone trabeculae and simultaneous new bone formation. In the diaphyseal bone marrow of affected long bones, the epitheloid cell granulomas appear to induce the endosteal new bone formation.

Transglycosylation of naringin by Bacillus stearothermophilusMaltogenic amylase to give glycosylated naringin.

Naringin, a bitter compound in citrus fruits, was transglycosylated by Bacillus stearothermophilus maltogenic amylase reaction with maltotriose to give a series of mono-, di-, and triglycosylnaringins. Glycosylation products of naringin were observed by TLC and HPLC. The major glycosylation product was purified by using a Sephadex LH-20 column. The sturcture was determined by using MALDI-TOF MS, methylation analysis, and (1)H and (13)C NMR. The major transglycosylation product was maltosylnaringin, in which the maltose unit was attached by an alpha-1-->6 glycosidic linkage to the D-glucose moiety of naringin. This product was 250 times more soluble in water and 10 times less bitter than naringin.

Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside.

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.

Development and sexual maturation of Taenia crassiceps (Cestoda) in the golden hamster.

The development of cysticerci of Taenia crassiceps to mature tapeworms in the intestinal tract of cortisone-treated and untreated golden hamsters is described. The development of the worms in untreated hamsters is as follows. Maturation and branching of the uterus and the presence of ova in the uterus were observed on day 15 postinfection (PI). By day 21 PI, fertilization had taken place, as evidenced by the presence of sperm in the seminal receptacle. On day 28 PI, shelled eggs were observed in feces. The rate of development of the worms in cortisone-treated hamsters was similar to that observed in untreated hamsters. More worm recovery and a longer period of worm survival were seen in the cortisone-treated than in the untreated hamsters. Thus, both cortisone-treated and untreated golden hamsters can produce normal gravid individuals of T. crassiceps.

Lysosomal enzymes in tear fluids from patients with ocular diseases.

Lysosomal enzyme activities in the tear fluids were determined in patients with ocular diseases. Acid phosphatase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, and beta-D-mannosidase activities were almost the same among the tear fluids from patients with myopia, rhegmatogenous retinal detachment, simple diabetic retinopathy and pigmentary retinal dystrophy. In contrast, the activities were lower in the affected eyes of patients with herpetic keratitis and vernal conjunctivitis than in the fellow normal eyes. It is possible that the lysosomal enzyme activities in the tears may be closely related to the condition of the external eye disease.