Subunit structure of isomeric chondroitin sulfates from normal human urine

Two temperature-sensitive mutants of Chlamydomonas reinhardii Dangeard which are defective in protein synthesis were examined. Both show breakdown of their polysomes at the restrictive temperature into monosomes which do not contain fragments of mRNA. Many of the ribosomes still contain nascent peptides able to react with puromycin. The polysome breakdown involves only cytoplasmic (80S) ribosomes and is prevented or reversed when ribosome translocation is inhibited with cyloheximide.     

Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma

Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.     

Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography

Biological samples have a high dielectric constant that can shorten RF wavelengths by a factor of 8 relative to the vacuum. At high field strengths, finite wavelength effects within larger samples are the dominant cause of RF field nonuniformity. A coil design is presented that can reduce and even eliminate this inhomogeneity; 4-T images in phantoms and in the head of a normal volunteer are presented, which demonstrate improved homogeneity relative to a standard coil. This coil design should aid in realizing the potential advantages of imaging large samples at high field strengths.     

Separation and quantification by ion-association capillary zone electrophoresis of unsaturated disaccharide units of chondroitin sulfates and oligosaccharides derived from hyaluronan

An analytical method has been developed for assay of unsaturated disaccharides of chondroitin sulfates and of oligosaccharides (tetra- and hexasaccharides) of hyaluronan, using ion-association capillary zone electrophoresis. Samples were applied at the anode (the usual polarity), using a borate buffer modified by an ion-pairing reagent, tetrabutylammonium (TBA) phosphate, and the effect of the concentration of the ion-pairing reagent on various electrophoretic parameters (electroosmotic flow, electrophoretic mobility of products, capacity factors) was observed. Increasing concentrations of the reagent led to a decrease of zeta potential, probably due to specific adsorption of the quaternary ammonium ion onto the capillary wall. The authors propose a mechanism of separation, in which anionic borate complexes are formed and interact with TBA ion inside the capillary tube. The capillary electrophoretic system described is potentially a powerful method for specific measurement of the concentrations in joint tissues of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronan, whose relative abundances may vary in various diseases or after local treatments with, for example, antiinflammatory drugs, chondroprotective agents, or orthopedic implants.     

Determinants of attention during impression formation.

Subjects in an impression formation task were timed as they read a list of behaviors attributed to a fictional target person. Reading times were longer for early behaviors than for behaviors later in the list (Experiment 1), and longer for behaviors inconsistent with the target's personality (Experiment 2). Moreover, inconsistent behaviors were studied longer when they occurred late in the list during on-line impression formation. These results provide direct support for an attentional account of the primacy effect in impression formation and for the recall advantage for inconsistent behaviors in person memory (Anderson & Hubert, 1963). An impression-sensitivity hypothesis is proposed that predicts that the salience of inconsistent information depends upon the state of an evolving impression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Human osteoclast formation and activity in vitro: effects of alendronate

A 10-center cooperative clinical study with a new formulation of epirubicin hydrochloride injectable solution (Epirubicin-RTU) was conducted in patients with breast cancer. One course of treatment consisted of one intravenous administration of Epirubicin-RTU at the dose of 60 mg/m2 followed by a 3-week drug-free interval and concomitant daily administration of oral cyclophosphamide at 100 mg/day during the period between Days 1 through 14. At least, two courses of treatment were given. Among 20 registered cases, all 20 cases were eligible and 16 cases completed the whole course of the study. In 16 completers, PR was observed in 5 cases, indicating the efficacy rate of 31.3% (5/16).. No local irritation was observed at the injection sites. Adverse reactions frequently observed were leukopenia, neutropenia, anorexia, alopecia, and nausea/vomiting, which were all reversible and tolerable. From the above results, Adverse reactions both locally and systemically were tolerable. Intravenous administration of Epirubicin-RTU was considered to be useful for the treatment of breast cancer.     

Plasmodium falciparum: enhanced gametocyte formation in vitro in reticulocyte-rich blood

Concentrates of late stage parasites of the gametocyte-forming clone HB-3 were mixed with blood rich in reticulocytes from anemic patients, or with normal control blood, and kept under culture conditions for 4 days. Significantly more gametocytes were always formed in the reticulocyte-rich blood than in the control. This was true whether the anemic blood supported a larger asexual parasitemia than the control, or a lower one, or the same and without regard to the cause of the anemia. Gametocytes as a percentage of asexual forms were up to 10 times higher in reticulocyte-rich blood than in normal blood.     

Suppression of pannus-like extension of synovial cells by lipid-derivatized chondroitin sulphate: in vitro and in vivo studies using Escherichia coli-induced arthritic rabbits

In rheumatoid arthritis, pannus formation resulting from synovial inflammation is a major factor in cartilage destruction. The ability of arthritic synovial cells to undergo pannus formation depends upon their initial adhesion to the partially deformed cartilage surfaces. Our recent studies using various lipid-derivatized glycosaminoglycans have revealed a preeminent inhibitory activity of phosphatidyl ethanol amine-derivatized chondroitin sulphate (CS-PE) toward cell-matrix adhesion. Here we evaluate whether CS-PE may protect articular cartilage from pannus extension in different in vitro and in vivo model systems using Escherichia coli 0:14-induced arthritis in rabbits and the articular cartilage explants, synovial tissues, and synovial cells obtained from them. These studies showed that CS-PE suppressed the in vivo pannus-like extension on cartilage surfaces, as well as the in vitro extension of the synovial cell layer on both CS-PE treated culture plates and cartilage explants. The results suggest that native chondroitin sulphate proteoglycans in the surface of normal articular cartilage play an important role in protecting the tissues from pannus extension and that the CS-PE immobilized onto partially eroded cartilage can mimic the inhibitory action of native chondroitin sulphate proteoglycans.     

Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.     

Progesterone formation and metabolism by rabbit placenta in vitro

4-14C-Progesterone and 4-14C-pregnenolone are metabolized in vitro by rabbit placenta, at day 15 and 28 of gestation, exclusively to compounds reduced in ring A (5beta) and at carbon 3 and 20.     

柠檬酸铅在柠檬酸钠溶液中溶解行为

通过氧化铅与柠檬酸反应制备了柠檬酸铅,考察了溶解时间、溶解温度、柠檬酸钠浓度和柠檬酸加入量对柠檬酸铅在柠檬酸钠溶液中溶解率的影响.结果表明：温度、柠檬酸钠浓度及柠檬酸加入量是主要影响因素,升高温度和提高柠檬酸钠浓度可显著提高柠檬酸铅溶解率;温度和溶解率呈正线性关系,拟合的线性方程为Y=0.76+0.63T;加入柠檬酸则对柠檬酸铅溶解有抑制作用.     

Mechanism of the citrate transporters in carbohydrate and citrate cometabolism in Lactococcus and Leuconostoc species

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178-2185, 1996). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis. The cloned gene was expressed in a Lactococcus lactis Cit- strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.     

Use of endometrial measurement as an exclusion criterion for in vitro fertilization using clomiphene citrate

OBJECTIVE: Previous reports have indicated an association between endometrial development and pregnancy outcome for patients treated with clomiphene citrate (CC) in conjunction with intrauterine insemination or intercourse. We expanded the use of CC for ovulation induction in association with in vitro fertilization (IVF). This study was designed to determine if endometrial thickness should be used as an inclusion or exclusion criterion for CC-IVF. STUDY DESIGN: One hundred twenty-eight patients were enrolled in an ovulation-induction regimen using CC for expected IVF-ET between January 1992 and December 1992. A total of 81 patients met inclusion criteria for CC-IVF and had endometrial measurement performed prior to human chorionic gonadotropin administration. Patients were categorized on the basis of endometrial measurement as follows: (A) > 4 - < 7 mm, (B) > or = 7 - < or = 10 mm, and (C) > 10 mm. Standard IVF was performed, and pregnancy rates for each category were evaluated. RESULTS: A total of 23 pregnancies (28% per retrieval) were established. Pregnancy rates were not different by category (P > .10, Fisher's Exact Test): (A) 3/15 (20%), (B) 13/41 (32%), and (C) 7/25 (28%). CONCLUSION: These data suggest that for CC-IVF. endometrial measurement should not be used as an exclusion criterion since pregnancies occurred at comparable frequencies in all the groups.     

V(D)J recombination: in vitro coding joint formation

Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion- and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.     

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.     

Oxidation of alkaline earth sulfides to sulfates: Thermodynamic aspects

The emf of the galvanic cell, Pt, Ni + NiO/(CaO) ZrO₂/MS + MSO₄, Ir, Pt, where M is calcium, strontium, or barium, has been measured in the temperature range 850 to 1100 K. From these measurements the Gibbs’ energy changes for the oxidation of sulfides of alkaline earth metals to their respective sulfates have been calculated. The results are compared with available thermodynamic data in the literature. The agreement varies from ±2 kJ for the strontium system to ±20 kJ in the case of barium. Trends in the stabilities of alkaline earth sulfates are discussed in relation to the properties of the cationic species involved.     

Laminin inhibits Abeta42 fibril formation in vitro

The mixed leukocyte reaction (MLR) is an in vitro test commonly performed in a serum-containing medium (SCM), and used to study allorecognition and cellular immunity accompanied by cytokine release. We investigated the possibility of performing the MLR test in serum-free media (SFM) by comparing human leukocyte proliferation and cytokine release in MLRs performed in SFM and SCM. Of the four SFM tested, only Biotarget- was as effective as SCM in supporting leukocyte proliferation and IL-2 secretion. Both phenomena were observed only in allogeneic combinations. The levels of IL-1, IL-6, and TNFalpha in allogeneic MLR combinations in SFM were half those in SCM cultures; the kinetics of their release were the same. With the exception of IL-2, a high degree of spontaneous release of the other three cytokines analyzed was observed in responder cells, in irradiated stimulator cells, and in autologous combinations cultured in both SCM and SFM. It appears that unlike IL-2, the cytokines IL-1, IL-6, and TNFalpha are nonspecifically produced in MLR and cannot serve as sensitive indices of HLA disparity.     

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.