黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Linkage of the gene for an X-linked mental retardation disorder to a hypervariable (AGAT)n repeat motif within the human hypoxanthine phosphoribosyltransferase (HPRT) locus (Xq26).

We recently reported a new X-linked mental retardation (XLMR) disorder in a four-generation family of Dutch descent. Features included Dandy-Walker malformation, basal ganglia disease, and seizures. Twenty-six family members, including two living affected males and two obligate carriers, were available for study. No evidence of linkage was observed between the disease locus and RFLPs from several X-chromosome regions, including Xp21-p22 (13 markers), proximal Xq (four markers), and Xq28 (three markers). However, a new hypervariable short tandem repeat (STR) within the HPRT gene at Xq26 showed positive linkage to the disease locus, with a maximum lod score of 2.19 at a recombination fraction of 0. A second hypervariable marker in Xq26, the dinucleotide repeat XL90A3 (DXS425), showed a lod score of .84 at a recombination fraction of .11. Both the HPRT and DXS425 markers were typed in 40 CEPH families, and subsequent multipoint linkage analysis showed the following order: Xcen-DXS425-(HPRT,XLMR)-F9-qter. HPRT and these flanking markers are therefore useful for carrier detection and prenatal diagnosis in this family. This study illustrates that hypervariable STRs will be powerful tools for linkage analysis and genetic diagnosis, particularly when relatively small families are involved.     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages

We have measured cytosolic free calcium levels ([Ca++]i) in individual macrophages during the phagocytosis of single zymosan particles. We report here that the contact of a macrophage with zymosan results in a rapid transient elevation of [Ca++]i. Each [Ca++]i pulse is symmetrical lasting for up to 30 seconds. In contrast, macrophage spreading is associated with repetitive [Ca++]i spiking occurring in salvos of up to four smaller spikes, each lasting for between 8 and 18 seconds. These qualitative and kinetic differences might suggest that the role of [Ca++]i in phagocytosis is distinct from its role in spreading.     

Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation.

Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L' conformations give rise to two new conformations, R' and R', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom

Arietin, an Arg-Gly-Asp containing peptide from venom of Bitis arietans, inhibited aggregation of platelets stimulated by a variety of agonists with a similar IC50, 1.3-2.7.10(-7) M. It blocked aggregation through the interference of fibrinogen binding to fibrinogen receptors on platelet surface. In this paper, we further demonstrated that arietin had no significant effect on the intracellular mobilization of Ca2+ in Quin2-AM-loaded platelets stimulated by thrombin. It inhibited 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50, 1.1.10(-7) M). 125I-arietin bound to unstimulated, ADP-stimulated and elastase-treated platelets in a saturable manner and its Kd values were estimated to be 3.4.10(-7), 3.4.10(-8) and 6.5.10(-8) M, respectively, while the corresponding binding sites were 46,904, 48,958 and 34,817 per platelet, respectively. Arg-Gly-Asp-Ser (RGDS) inhibited 125I-arietin binding to ADP-stimulated platelets in a competitive manner. RGD-containing peptides, including trigramin and rhodostomin, EDTA and monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex, inhibited 125I-arietin binding to ADP-stimulated platelets, indicating that the binding sites of arietin appear to be located at or near glycoprotein IIb-IIIa complex. In conclusion, arietin and other RGD-containing trigramin-like peptides preferentially bind to the fibrinogen receptors associated with glycoprotein IIb-IIIa complex of the activated platelets, thus leading to the blockade of fibrinogen binding to its receptors and subsequent aggregation. The presence of RGD of arietin is essential for the expression of its biological activity. Its binding sites are overlapped with those of trigramin, rhodostomin and the monoclonal antibody, 7E3.     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Biological activities of melanotropic peptide fatty acid conjugates.

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the alpha-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a "creeping" potency in the lizard skin bioassay-that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10-100 times more active than alpha-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Inspection and feeding of larvae by worker honey bees (Hymenoptera: Apidae): Effect of starvation and food quantity

Honey bee larvae are frequently inspected and, sometimes, provided with food by adult workers, but the stimuli that elicit the important task of food provisioning have never been investigated. Larvae with their food experimentally deprived received more frequent inspection and feeding visits from nurse bees than normally fed larvae, suggesting that there could be a hunger signal. Food-deprived larvae with artificially supplied larval food received the same rate of feeding visits from nurse bees as did normally fed larvae but still received more inspection visits. These results suggest that stimuli eliciting feeding are different from those for inspection. They also support the hypothesis that worker bees deposit food in a larval cell only when the quantity of food is below a certain minimum threshold that is perceived during larval inspections. A model is presented regarding the stimuli from larvae that result in worker feeding behavior.     

The emergence of sense organs in the wing disc of Drosophila

We have examined the origin of a set of precisely located sense organs in the notum and wing of Drosophila, in transformant flies where lacZ is expressed in the progenitor cells of the sense organs (the sensory mother cells) and in their progeny. Here we describe the temporal pattern of appearance and divisions of the sensory mother cells that will form the eleven macrochaetes and the two trichoid sensilla of the notum, and five campaniform sensilla on the wing blade. The complete pattern of sensory mother cells develops in a strict sequence that extends over most of the third larval instar and the first 10 h after puparium formation. The delay between the onset of lacZ expression and the first differentiative division ranges from 30 h, in the case of the earliest mother cells, to 2 h for the latest mother cells. The first division shows a preferential orientation which is also specific for each sensory mother cell. Up to this stage, there is no marked difference between the three types of mechanosensory organs.