Regulation of prolactin gene expression during early pregnancy in rats

We have shown that administration of estrogen which increases prolactin (PRL) synthesis in the rat may be mediated by an increase in poly [adenosine diphosphate ribose (ADP-ribose)] synthesis. Present investigation was attempted to study whether poly (ADP-ribose) synthesis is involved in rat PRL gene expression during early pregnancy. Anterior pituitaries were obtained on days 0, 2, 4, 6, 8, 10 and 12 of pregnancy (group C). Another group of pregnant rats was given nicotinamide, an inhibitor of poly (ADP-ribose) synthesis twice a day intra-peritoneally from day 0 to the day of sacrifice (group N). Serum estradiol (E2) concentration was determined by radioimmunoassay. PRL mRNA was measured by cytoplasmic dot hybridization using 32P-labeled cDNA. Poly (ADP-ribose) synthesis was assessed by incubating purified nuclei with 14C-nicotinamide adenine dinucleotide. The serum concentration of E2 increased between days 2 and 4, and on day 6 it decreased to the level of day 0. It remained low until day 12. No difference in the serum E2 level was observed in groups C and N. In group C, PRL mRNA increased from day 2 and remained high until day 8. In group C, poly (ADP-ribose) synthesis increased between days 2 and 4, decreased on day 6 to the level of day 0, and thereafter gradually increased until day 10. Administration of nicotinamide abolished the increase in poly (ADP-ribose) synthesis observed in group C during early pregnancy. In group N, the increase in PRL mRNA was completely suppressed. It is suggested that the increase in PRL mRNA in early pregnancy may be mediated by increased poly (ADP-ribose) synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.     

Purification, Molecular Cloning, and Enzymatic Properties of a Family 12 Endoglucanase (EG-II) from Fomitopsis palustris: Role of EG-II in Larch Holocellulose Hydrolysis

A family 12 endoglucanase with a molecular mass of 23,926 Da (EG-II) from the brown-rot basidiomycete Fomitopsis palustris was purified and characterized. One of the roles of EG-II in wood degradation is thought to be to loosen the polysaccharide network in cell walls by disentangling hemicelluloses that are associated with cellulose.     

Relationship among coronary plaque compliance, coronary risk factors and tissue characteristics evaluated by integrated backscatter intravascular ultrasound

ABSTRACT: BACKGROUND: The purpose of the present study was to evaluate the mechanical properties of coronary plaques and plaque behavior, and to elucidate the relationship among tissue characteristics of coronary plaques, mechanical properties and coronary risk factors using integrated backscatter intravascular ultrasound (IB-IVUS). Methods: Non-targeted plaques with moderate stenosis (plaque burden at the minimal lumen site: 50-70%) located proximal to the site of the percutaneous coronary intervention target lesions were evaluated by IB-IVUS. Thirty-six plaques (less calcified group: an arc of calcification [less than or equal to]10) in 36 patients and 22 plaques (moderately calcified group: 10< an arc of calcification [less than or equal to]60) in 22 patients were evaluated. External elastic membrane volume (EEMV) compliance, lumen volume (LV) compliance, plaque volume (PV) response (difference between PV in systole and diastole), EEM area stiffness index were measured at the minimal lumen site. Relative lipid volume (lipid volume/internal elastic membrane volume) was calculated by IB-IVUS. Results: In the less calcified group, there was a significant correlation between EEMV compliance and the relative lipid volume (r=0.456, p=0.005). There was a significant inverse correlation between EEM area stiffness index and the relative lipid volume (p=0.032, r =-0.358). The LV compliance and EEM area stiffness index were significantly different in the diabetes mellitus (DM) group than in the non-DM group (1.32 +/- 1.49 vs. 2.47 +/- 1.79 %/10 mmHg, p =0.014 and 28.3 +/- 26.0 vs. 15.7 +/- 17.2, p =0.020). The EEMV compliance and EEM area stiffness index were significantly different in the hypertension (HTN) group than in the non-HTN group (0.77 +/- 0.68 vs. 1.57 +/- 0.95 %/10 mmHg, p =0.012 and 26.5 +/- 24.3 vs. 13.0 +/- 16.7, p =0.020). These relationships were not seen in the moderately calcified group. Conclusion: The present study provided new findings that there was a significant correlation between mechanical properties and tissue characteristics of coronary arteries. In addition, our results suggested that the EEMV compliance and the LV compliance were independent and the compliance was significantly impaired in the patients with DM and/or HTN. Assessment of coronary mechanical properties during PCI may provide us with useful information regarding the risk stratification of patients with coronary heart disease.     

Structure of the F‐spondin domain of mindin,an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.     

Load-dependent regulation of neuromuscular system

Roles of gravitational loading, sarcomere length, and/or tension development on the electromyogram (EMG) of soleus and afferent neurogram recorded at the L5 segmental level of spinal cord were investigated during parabolic flight of a jet airplane or hindlimb suspension in conscious rats. Both EMG and neurogram levels were increased when the gravity levels were elevated from 1-G to 2-G during the parabolic flight. They were decreased when the hindlimbs were unloaded by exposure to actual microgravity or by suspension. These phenomena were related to passive shortening of muscle fibers and/or sarcomeres. Unloading-related decrease in sarcomere length was greater at the central rather than the proximal and distal regions of fibers. These activities and tension development were not detected when the mean sarcomere length was less than 2.03 micrometers. It is suggested that load-dependent regulation of neuromuscular system is related to the tension development which is influenced by sarcomere length.     

Differential expression of fucosyl GM1 and a disialoganglioside with a NeuAc alpha 2-6GalNAc linkage (GD1e) in various rat ascites hepatoma cells

A distinct difference in ganglioside composition among various rat ascites hepatomas and Yoshida sarcoma was observed on TLC-immunostaining with anti-fucosyl GM1 antibody, and chemical and enzymatic analyses. Yoshida sarcoma and ascites hepatomas, AH13, AH66F and AH66, but not the other 9 tumor cell lines investigated, specifically contained a disialoganglioside, NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1ceramide (GD1e), whereas the 9 ascites hepatoma cells without GD1e contained fucosyl GM1. The differential expression of fucosyl GM1 and GD1e in various tumor cell lines indicates that different cell lineages express distinct metabolic pathways for gangliosides, and that the gangliosides are useful markers for distinguishing tumor cell lines.     

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.     

A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.