Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.     

Materials for the fungus flora of Japan (49)

Two interesting microfungi are described as new to Japan:Talaromyces galapagensis (anam.Penicillium galapagense), isolated from soil in Shizuoka; andPenicillium megasporum, isolated from marine sludge in Nagasaki. Some observations are recorded, particularly on ascomatal initials ofT. galapagensis, which are similar to those described forTalaromyces flavus.(48): Kaneko, S., Mycoscience 36: 359–360, 1995.     

Talaromyces lagunensis,a new species from Philippine soil

A new species ofTalaromyces (Ascomycetes; Trichocomaceae) with aPenicillium anamorph,T. lagunensis, is described and illustrated. This fungus is characterized by its extremely restricted growh on Czapek-yeast extract agar, light yellow to light orange ascomata with a telaperidium, catenate, pyriform or ellipsoidal asci, ellipsoidal or subglobose ascospores with a microtuberculate wall, short conidiophores with an irregular, mostly monoverticillate to biverticillate penicillus, and subglobose to ovoid conidia. The holotype was isolated from forest soil in the Philippines.     

Change in Hair Cycle and Hair Length in Nude Mice by Administration of Deuterium Oxide

D₂O increased hair length in Balb/c nu/nu (nude) mice in our previous study although it has an antimitotic effect in cells. To investigate the mechanism of the effect on the hair length, we examined the change by the administration of D₂O in the duration of the hair cycle and the proliferating activity of the hair matrix in relation with hair length in nude mice. The results showed that 20 or 30% D₂O administration did not change the gross structure of the hairs, the proliferative activity and keratinization of the hair matrix cells, but elongated the hair cycle. The duration of the hair cycle increased by the administration of D₂O in a dose-dependent manner over the examined range and these effects were reversible by discontinuation of D₂O. The change in the hair length correlated with the change in the hair-existing phase particularly. We also showed that the mast cell density in the skin, which is related to the hair cycle, increased in the deuterated mice at anagen VI stage which nearly corresponds to the hair-existing phase. The increase in the mast cell density may be related to the increase in the hair cycle duration. These findings indicate that the increase in hair length may be due to the increase in the duration of the hair cycle, in particular, an increase in the hair-existing phase. This study thus suggests that D₂O slows not only short-term cycles such as circadian clock or ultradian clock, but also the hair cycle which is a long-term cycle.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Ca2+-induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes

The effects of Ca²⁺ on phosphatidic acid-phosphatidylcholine membranes have been studied using phospholipid spin labels. ESR spectra of spin-labeled phosphatidic acid-phosphatidylcholine membranes and phosphatidic acid-spin-labeled phosphatidylcholine membranes are exchange-broadened immediately upon addition of CaCl₂. These changes directly and conclusively indicate Ca²⁺-induced clustering of spin-labeled phosphatidylcholine and aggregation of spin-labeled phosphatidic acid bridged by Ca²⁺-chelation in the binary phopholipid membranes. In the Ca²⁺-chelated aggregates, the motions of the alkyl chains of phosphatidic acid are greatly reduced and the lipid molecules are more closely packed. The clusters and aggregates are formed in patches and the sizes are dependent on the fractions. Ba²⁺ and Sr²⁺ induce the lateral phase separations to the same extent as Ca²⁺. Mg²⁺ is also effective but to a lesser extent. In acid solutions (pH 5.5), the Ca²⁺-induced lateral phase separations are of slightly lesser extent than in alkaline solution (pH 7.9). These results are compared with those for phosphatidylserine-phosphatidylcholine membranes reported previously and necessary conditions for the lateral phase separations are discussed.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.