Simian retrovirus-D serotype 1 (SRV-1) envelope glycoproteins gp70 and gp20: expression in yeast cells and identification of specific antibodies in sera from monkeys that recovered from SRV-1 infection.

The gp70 and transmembrane gp20 envelope proteins of simian retrovirus-D serotype 1 (SRV-1) were expressed in Saccharomyces cerevisiae as fusion proteins with human superoxide dismutase (SOD). Expression of the SOD-gp70 and SOD-gp20 sequences yielded fusion proteins of 52 and 29 kilodaltons, respectively. The yeast-expressed SRV-1 envelope proteins were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in the sera of rhesus macaques that recovered from SRV-1. Sera from 47 of 49 such monkeys tested positive for antibodies to the SOD-gp70 fusion protein, while 45 of 49 reacted positively to SOD-gp20. None of 26 SRV-1-nonexposed monkeys tested positive in either ELISA. Monkeys immunized with the recombinant SRV-1 gp20 and gp70 proteins made good ELISA and Western blot (immunoblot) antibodies to whole SRV-1. This antibody was not neutralizing in vitro, however.     

Monoclonal antibodies to three phospholipase C isozymes from bovine brain

Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denatured 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II, and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II, and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II, and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II, and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes.     

The ontogeny of the vascular cambium inGinkgo biloba roots

Observation was made on early ontogeny of vascular cambium in the developing root ofGinkgo biloba L. After completion of root elongation, the vascular meristem gradually acquires cambial characteristics. Strips of the periclinal division of cells in transverse section are observed on the inner side of phloem when the primary xylem and phloem in the stele have been established. The strips are united into a continuous layer between phloem and xylem. In tangenital section, the procambium shows a homogeneous structure, which is initially composed of short cells with transverse end walls and subsequently, of long cells with tapering ends. Then, the procambium is organized into two systems of cells; axial strands of short cells with transverse end walls resulting from the sporadic transverse divisions of long cells, and long cells with tapering ends. Still later, the short cells are divided frequently in a trasverse plane exhibiting one or a few cells in width and several decades of cells in height, while the long cells are elongated. The frequency of transverse divisions of the short cells decreases in subsequent stages. Eventually, the short cells in axial strands are vertically separated from one another by the elongation of neighboring long cells and by the decrease in the frequency of transverse divisions of short cells themselves. Cambial initials occur in two forms; ray initials a few cells in height and one cell in width derived from the short cells, and fusiform initials with tapering ends derived from the long cells.     

The effect of high pressure sodium vapor lamps on the rat

The effect of high pressure sodium vapor lamps and daylight-simulating fluorescent lights on the growth, hematology, and behavior of Sprague-Dawley rats has been investigated. Rats weaned under the sodium vapor lamps had slightly heavier adrenals than those exposed to the daylight lamps when the two lighting systems were equalized either for total irradiance or for scotopic illuminance. However, no differences were observed in the tail-flick response, hot plate response, or swimming endurance of rats housed under the two lighting conditions. No consistent differences were seen in the hemoglobin, red and white cell count, hematocrit, and mean cell volume between rats weaned under the high pressure sodium vapor lamps and daylight-simulating fluorescent lights.     

The development of a technique for the morphometric analysis of invasion in cancer

We describe tests of the feasibility of a reconstructive technique to discriminate between expansive growth and active cell movement in the invasion of tissues by cancer cells. The densities of cancer cells in 2210 microns2 (grid) squares of standard 6 microns fixed, stained histologic sections of a nodule and an invasive cutaneous melanoma were determined, and density maps of the tumors constructed. An abrupt transition from saturation density to zero cell density was observed at the advancing edge (towards the stratum corneum) of the tumor nodule which was consistent with a model for expansion by growth (vis a tergo). In contrast, at the advancing edge of the invasive tumor, the transition from saturation to zero density (towards the subcutaneous tissues) occurred more gradually, over approximately 400 mum, which was consistent with a model for invasion by active movement of melanoma cells. The occurrence of statistically significant "high density regions" near to the advancing edge of the invasive tumor is consistent with an invasive pattern of active movement followed by focal proliferation of the cancer cells, in a repetitious manner. It therefore appears feasible to make kinetic reconstructions of some of the events in invasion, from static quantitative observations.     

Typing of Nontypable Staphylococci by Lysogeny

Strains of coagulase-positive staphylococci which were nontypable with the routine typing set of phages could be typed by lysogeny with phage-propagating strains as indicators and with ultraviolet induction. About 10% of the strains could be typed without induction. About 36% of them could be typed by this method when ultraviolet irradiation was used as an inducing agent. The phage groups from which the majority of the nontypable staphylococci originated were easily identified by this method of typing.     

Lack of antinociceptive cross-tolerance between intracerebroventricularly administered beta-endorphin and morphine or DPDPE in mice

Antinociceptive tolerance and cross-tolerance to intracerebroventricular (i.c.v.) beta-endorphin, morphine, and DPDPE (D-Pen2-D-Pen5-enkephalin) induced by a prior i.c.v. administration of beta-endorphin, morphine and DPDPE, respectively, were studied in mice. Acute tolerance was induced by i.c.v. pretreatment with beta-endorphin (0.58 nmol), morphine (6 nmol) and DPDPE (31 nmol) for 120, 180 and 75 min, respectively. Various doses of beta-endorphin, morphine or DPDPE were then injected. The tail-flick and hot-plate tests were used as antinociceptive tests. Pretreatment of mice with beta-endorphin i.c.v. reduced inhibition of the tail-flick and hot-plate responses to i.c.v. administered beta-endorphin, but not morphine and DPDPE. Pretreatment of mice with morphine i.c.v. reduced inhibition of the tail-flick and hot-plate responses to morphine but not beta-endorphin. Pretreatment of mice with DPDPE reduced inhibition of the tail-flick and hot-plate responses to DPDPE but not beta-endorphin. The results indicate that one injection of beta-endorphin, morphine or DPDPE induces acute antinociceptive tolerance to its own distinctive opioid receptor and does not induce cross-tolerance to other opioid agonists with different opioid receptor specificities. The data support the hypothesis that beta-endorphin, morphine and DPDPE produce antinociception by stimulating specific epsilon, mu- and delta-opioid receptors, respectively.     

Fluorescence probes in biochemistry: an examination of the non-fluorescent behavior of dansylamide by photoacoustic calorimetry.

Photoacoustic calorimetry is shown to be a simple, precise, and accurate method for the quantification of the photophysics of a fluorescence probe, e.g., dansylamide, in a variety of solvents. This technique, which is described in detail, provides a direct measurement of the energy that is released nonradiatively following photostimulation, and can therefore be used to indirectly determine the amount of energy released via luminescent pathways. Photoacoustic calorimetry combined with established absorption and fluorescence methodologies provides a complete arsenal for characterizing the photophysical properties of many systems. Comparison of the photoacoustic signal for dansylamide versus standard compounds (ferrocene, tetraphenylethylene, 8-anilinonaphthalene-1-sulfonate, and/or 5,5'-dithiobis(2-nitrobenzoic acid) in 12 different solvents gave fh values (fraction of each absorbed 337.1-nm photon returned as heat) from a low of 0.530 in 1,4-dioxane to a high of 0.973 in water. The trend noted with solvent polarity is different and more revealing than that determined by the more classical approach of examining either the wavelength of the emission maximum or the fluorescence quantum yield.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996