Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Genistein binding protein targets in dental pathogens

Oral pathogens have created a menace in recent years due to biofilm formation and antimicrobial drug resistance. The current treatment strategy works well with antibiotics. However, constant use of antibiotics creates a selective pressure, which increases adaptability of the pathogens. Therefore, it is of interest to analyze the potential targets of genistein in dental pathogens using computer aided prediction tools.     

Differential inhibition of human immunodeficiency virus type 1 fusion, gp120 binding, and CC-chemokine activity by monoclonal antibodies to CCR5

The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.     

Analysis of superfamily specific profile-profile recognition accuracy

Background  

Annotation of sequences that share little similarity to sequences of known function remains a major obstacle in genome annotation. Some of the best methods of detecting remote relationships between protein sequences are based on matching sequence profiles. We analyse the superfamily specific performance of sequence profile-profile matching. Our benchmark consists of a set of 16 protein superfamilies that are highly diverse at the sequence level. We relate the performance to the number of sequences in the profiles, the profile diversity and the extent of structural conservation in the superfamily.     

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.     

CCR5 and CXCR4 usage by non-clade B human immunodeficiency virus type 1 primary isolates

CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.