Detection of HIV-1 DNA in cells and tissue by fluorescent in situ 5'-nuclease assay (FISNA).

The critical aspects of successful in situ amplification include fixation, permeabilization, amplification and detection. We address these aspects and present a novel detection scheme that eliminates hybridization following amplification. We use the 5'-nuclease activity of Taq polymerase to cleave in situ a 5'-reporter dye from an oligonucleotide probe which hybridizes to the target amplicon during amplification. The 5'-reporter dye is disassociated from the 3'-quenching dye and remains localized by charge interactions. In addition, we describe probe design constraints for 5'-nuclease assays both in solution and in situ. Using this technique, we show the sensitive and specific detection of HIV-1 DNA in cells lines and tissue from HIV-1-infected individuals.     

Identification of a new contingency-based response in honey bees (Apis mellifera) through revision of the proboscis extension conditioning paradigm

The two experiments reported present new information in the area of classical conditioning experiments with honey bees. Experiment 1 establishes a single unconditioned stimulus (US) technique as a preferred technique for conditioning of the proboscis extension response. Experiment 1 further identifies a new head turn response which occurs when the standard compound US technique is used. Experiment 2 demonstrates that the newly identified head turn response is contingency-based and provides important new response to the repertoire of honey bee learning experiments.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Electrophoretic Characteristics of Staphylococcal Hyaluronate Lyase

Partially purified staphylococcal hyaluronidase was studied with respect to its electrophoretic properties by the use of starch slurry or semisolid Noble agar as a matrix. The polarity of enzyme activity was found to be cathodal on starch and anodal on agar gel. The electrophoretic migration of the partially purified enzyme on starch, as a function of pH, suggested that the enzyme has an isoelectric point of pH 9.5 to 10.     

Antigen presentation to human T lymphocytes. II. Requirements for Mac-120+ macrophages and responsiveness to interleukin 2

To investigate mechanisms by which antigen, macrophages, and interleukin 2 (IL2) participate in the induction of secondary T-cell proliferative responses, trinitrophenyl (TNP) was presented in three distinct modes: (i) TNP-modified peripheral blood mononuclear cells (TNP-PBMC), (ii) TNP-PBMC cell sonicates, and (iii) TNP-ovalbumin (TNP-OVA). Stimulators were depleted of Mac-120+ macrophages using Mac-120 monoclonal antibody plus complement. TNP-Mac-120 macrophages stimulated primed T cells nearly as well as TNP-unfractionated macrophages (which were about 40% Mac-120+). In contrast, although greater than 70% DR+, Mac-120- macrophages plus either TNP-OVA or TNP-PBMC sonicate elicited minimal responses compared to unfractionated macrophages plus antigen. After 21-28 days of in vitro priming, macrophage-depleted T cells were not stimulated to proliferate by either IL2 alone or sonicates alone. IL2 plus TNP-PBMC sonicates, however, stimulated significant proliferation. Furthermore, this response was considerably greater than that to IL2 plus either TNP-T cell sonicates or TNP-mouse spleen sonicates. Thus, the Mac-120+ macrophage population may have an important antigen-presenting and/or accessory function in the stimulation of primed T cells by soluble or particulate antigen, although it is unnecessary for responses to intact TNP-Ia+ PBMC. In addition, the data suggest that Ia+ sonicates alone may suffice for induction of IL2 responsiveness, but not for endogenous IL2 production and subsequent proliferation by primed T cells.     

Triterpenes and sterols of Buxus sempervirens and local variations in their levels

The leaves of Buxus sempervirens L. contain sitosterol, stigmasterol, cycloartenol, lupeol, germanicol and β-amyrin in the free state. All of these compounds, except stigmasterol, were also found in the esters fraction, as were obtusifoliol, 24-methylenelophenol, and 24-ethylidenelophenol, The triterpene diois betulin and moradiol were isolated, the latter for the first time from a plant source. In nineteen Buxus samples from England, Wales and Scotland, the sterol compositions were quite similar while those of the triterpene monols varied considerably.     

Cytokinesis in HeLa: Post-telophase delay and microtubule-associated motility

Summary Phase-contrast microcinematography of cultured HeLa cells reveals that cell separation is considerably delayed after telophase. During this period of delay, the daughter cells lose their rounded morphology and become flattened against the substrate, as occurs in interphase. After two or more hours, the cells again become rounded while the thin intercellular bridge connecting them begins to elongate. This active elongation involves the migration of thickenings (waves) along the bridge from the midbody at its center toward either cell. Later, waves occur only on one side of the midbody as this half of the bridge alone continues to elongate. The arrival of waves at the cells is accompanied not only by discrete increases of length in that half of the bridge, but also by blebbing activity in that cell. Rupture of the bridge finally occurs just adjacent to the cell receiving these latter waves.Electron microscopic examination of cells in post-telophase delay has demonstrated a bundle of microtubules passing into either cell from the midbody in the center of the intercellular bridge. These microtubules are of constant length during bridge elongation; the cells are simply forced distally along the surface of the microtubule bundle. The waves themselves are found to contain microtubules just as straight as those in the rest of the bridge, so it is concluded that the force apparently generated here consists of the longitudinal translation of material along the surfaces of the rigid microtubules. It is pointed out that these forces may operate in the earlier phases of mitosis and in other systems of microtubule-associated motility. We also discuss the possible roles of post-telophase delay and of active bridge elongation in the organization of normal tissues.     

THE MECHANISM OF THE INFLAMMATORY PROCESS : III. ELECTROPHORETIC MIGRATION OF INERT PARTICLES AND BLOOD CELLS IN GELATIN SOLS AND GELS WITH REFERENCE TO LEUCOCYTE EMIGRATION THROUGH THE CAPILLARY WALL.

1. Quartz particles and certain other particles move cataphoretically in certain soft gelatin gels, with the same velocity as in the sol. The speed is a function of the true viscosity of the sol or gel, and it is See PDF for Structure apparently not altered in these soft gels by the presence of gel structure. It is proportional to the applied difference of potential. 2. This finding is compatible with the fact that certain sols undergo gelation with no increase of the true viscosity although a marked change in the apparent viscosity takes place. 3. Red cells in soft gelatin-serum gels show a distinct difference in behavior. They migrate through the sol or gel with a speed that is about twice as great as the leucocytes and quartz particles, which latter particles migrate with the same velocity. This ratio has been found to hold for serum and plasma. The absolute velocities are comparatively slightly decreased by the presence of the gel. 4. In more concentrated or stiffer gels, leucocytes, red cells and quartz particles all move at first with the same velocity. By producing mechanical softening of these gels (shearing from cataphoretic movement of the micells within the cell) the red cells presently resume their previous property of independent migration through the gel. 5. The movements of particles in gelatin gels produced by a magnetic force or the force of gravity are of a different nature than those movements produced by cataphoresis. 6. The mechanical nature of obstruction to the cataphoretic migration of leucocytes and red cells in fibrin gels is briefly described. 7. The correlation of cataphoresis of microscopic particles in gels with the order of magnitude and nature of the potential differences in the capillary wall, lends additional evidence to the theory that polymorphonuclear leucocyte emigration and migration are dependent upon these potential differences.     

ELECTROKINETIC PHENOMENA : XIII. A COMPARISON OF THE ISOELECTRIC POINTS OF DISSOLVED AND CRYSTALLINE AMINO ACIDS

1. Although the isoelectric points of dissolved cystine, tyrosine, and aspartic acid molecules lie at widely differing pH values, the isoelectric points of the surfaces of these substances in the crystalline state are all near pH 2.3. This was found to be true in solutions of hydrochloric acid and in acetate buffers of approximately constant ionic strength. 2. When suspended in gelatin, tyrosine and cystine crystals adsorb the protein and attain a surface identical in behavior with gelatin-coated quartz or collodion particles. 3. Aluminum ions at low concentrations reduce the electric mobilities of tyrosine crystals to zero in a manner analogous to their effect on other surfaces. 4. Alkyl benzene droplets also have their electric mobility reduced to zero at low pH values but, unlike the amino acids, a change in sign was never noticed. 5. The mobility of tyrosine crystals is independent of crystal length between 2–100µ. Below this size the mobilities are decreased. 6. These results are discussed in connection with the concept of the general definition of the isoelectric point and the behavior of certain insoluble proteins such as wool and silk fibroin.     

Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils.

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.