Diminishing adenovirus gene expression and viral replication by promoter replacement.

The adenovirus E4 promoter was replaced by a synthetic promoter composed of a minimal TATA box and five consensus 17-mer yeast GAL4-binding-site elements. The viral vectors, which also contained human factor IX (hFIX) cDNA driven by Rous sarcoma virus long terminal repeat in the E1 region, were then constructed and expanded in 293 cells permanently expressing GAL4/VP16 fusion protein. Viral replication and expression of adenovirus E4 genes and late genes (hexon and fiber) were evaluated in vitro in the human lung carcinoma cell line H1299. Viral replication and viral gene expression were dramatically reduced in the cells transduced by vectors with a replaced E4 promoter compared to the levels in the cells transduced by vectors with the wild-type E4 promoter. The levels of transgene (hFIX) expression remained similar between vectors with or without E4 promoter replacement. These results indicate that diminution of viral gene expression and viral replication is achievable by promoter replacement.     

Binocular depth perception mechanisms in tongue-projecting salamanders

Tongue-projecting salamanders (Bolitoglossini) combine extreme speed and high precision in prey capture. They possess all requirements for stereoscopic depth perception: frontally oriented eyes, a substantial amount of direct ipsilateral projection in addition to the contralateral one, and binocularly driven neurons. Extracellular recordings were made from retinal afferents in the tectum as well as from the somata of tectal neurons. RF-sizes of afferents and tectal neurons were determined, and the response properties of tectal neurons were tested under monocular and binocular conditions with stimuli of different size and velocity. While RF-sizes and response properties of binocular neurons during binocular and contralateral stimulation were similar, ipsilaterally stimulated neurons exhibited much smaller RFs, lower spike rates and different size preferences.Furthermore, the contralateral retinotectal projection from one eye and the ipsilateral from the other are in register. While retinal afferents are distributed linearly over the tectal surface, most tectal neurons are activated by a retinal area corresponding to the frontal visual field; this results in a magnification of this region. The two monocular receptive fields of binocular neurons exhibit zero disparities (horopter) at distances that coincide with the maximum reach of the tongue. We hypothesize that bolitoglossine salamanders (as well as amphibians in general) make use of two kinds of disparities: (1) between the maps in the left and right tectal hemisphere, coding for the lateral eccentricity of an object, and (2) between the ipsilateral and contralateral retinotectal map, coding for the distance. The presence of substantial direct ipsilateral afferents in bolitoglossine salamanders appears to be the basis for a fast computation of object distance, which is characteristic of these animals.Abbreviations Ax/Ay coordinates of a recorded afference - Nx/Ny coordinates of a recorded neuron - RF receptive field - RFc contralateral receptive field - RFi ipsilateral receptive field - RFx/RFy coordinates of a receptive field center - RGC retinal ganglion cell     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

An Artifact Of Liquid Emulsion Autoradiography

The localization of ³H-opiatcs in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ilial sections to dried photographic emulsion avoids this source of error.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Nippostrongylus brasiliensis: Peripheral leukocyte responses and correlation of basophils with blood histamine concentration during infection in rats

Rats infected on Day 0 with 3000 infective L₃ larvae of Nippostrongylus brasiliensis, and uninfected controls, were monitored daily through Day 23 postinfection for changes in peripheral leukocytes and blood histamine concentrations. A generalized leukocytosis was observed between Days 7 and 18, the period leading up to and immediately following the time of expulsion of adult worms from the small intestine. The total number of lymphocytes was elevated between Days 11 and 17 post-infection; however, there was no change in the percentage of lymphocytes relative to other white blood cell types. The total number and percentage of monocytes were no different from controls, with the exception of Day 5 postinfection. On that day, there was a significant elevation in the number (614/mm³ blood in infected rats, as compared to 160/mm³ blood in controls) and relative proportion (2.7% of total leukocytes in infected animals, compared to 0.8% in controls) of monocytes, coinciding with the termination of the pulmonary migration of larvae. A period of moderate neutrophilia occurred between Days 7 and 12, but this was not accompanied by any changes in the proportion of neutrophils. A biphasic eosinophil response was observed. An early elevation of eosinophils occurred between Days 3 and 5, corresponding to the period of larval migration through the lungs. A second period of eosinophilia began on Day 11, when worm expulsion was beginning, and continued through Day 19, i.e., beyond the period of worm expulsion. Basophilia was observed as early as Day 6 after infection, rising to a peak on Day 13 (6.8% of total leukocytes in the infected animals, as compared to 0.5% in controls), and declining thereafter, but remaining above control levels until termination of the experiment on Day 23. The histamine content of blood samples, as determined by an enzymic-isotopic assay, closely paralleled the development and decline of basophilia; histamine levels also peaked on Day 13 postinfection (422.5 pg histamine/mm³ blood in infected rats, compared to 66.0 pg histamine/mm³ blood in controls). As basophilia progressed during the course of infection, there was a decline in the amount of histamine per basophil. In uninfected rats and during the first week after infection, basophils contained about 1.5–2.0 pg histamine per cell. In the third week of infection, there was about 0.6 pg histamine per basophil. The time course of the basophilia suggests that these cells may be involved in the expression of immunity to N. brasiliensis.     

Zinc and insulin metabolism

A review of experimental studies of the effect of zinc nutrition on insulin metabolism is presented. In addition to a short introduction to the synthesis, secretion, and action of insulin, the effects of zinc deficiency—specifically on glucose tolerance, insulin secretion, insulin synthesis and storage, and on total insulin-like activity—are dealt with. The concentrations of zinc and chromium in serum, pancreas, and liver are compared to those of zinc-deficient animals and pair-fed controls. In contrast to pair-fed controls, zinc-deficient rats had unaltered proinsulin contents after glucose stimulation, but they showed a diminished glucose tolerance, lowered serum insulin content, and an elevated total insulin-like activity. The serum zinc concentration of the deficient animals was greatly reduced and did not change during glucose stimulation, whereas it rose in the case of the pair-fed controls. The serum chromium concentration increased in both groups in response to glucose stimulation. In the pancreas of the deficient animals, the zinc concentration was reduced 60% and it increased during the glucose tolerance test. In the liver there were no significant differences. The chromium concentrations were elevated in both the pancreas and liver of the zinc-deficient rats by 60 and 100%, respectively, and were not influenced by glucose injection. These studies show clearly that nutritional zinc deficiency influences insulin metabolism and action.     

High-affinity binding of lower-density lipoproteins to chicken oocyte membranes.

Oocyte membrane fragments bind specifically radioiodinated VLD lipoprotein (very-low density lipoprotein) and LD lipoprotein (low-density lipoprotein). Competitive binding assays showed 2-3 times more VLD lipoprotein than LD lipoprotein bound at 4 degrees C. Equilibrium-binding data revealed the presence of one class of non-interacting sites for VLD lipoprotein (kD 12 microgram/ml) and co-operative binding for LD lipoprotein. The binding of VLD lipoprotein showed a distinct pH maximum at 5.3, whereas an indistinct maximum at about pH 7.3 was observed for LD lipoprotein. Unlabelled VLD lipoprotein did compete with 125I-labelled LD lipoprotein binding, but unlabelled LD lipoprotein did not compete with 125I-labelled VLD lipoprotein binding. VLD lipoprotein binding was inhibited by HD lipoprotein (high-density lipoprotein), but not by lysozyme, collagen, poly-L-lysine or poly-L-arginine; LD lipoprotein binding was inhibited by lysozyme and collagen, but not by HD lipoprotein. On the basis of these studies, we suggest that: (1) VLD lipoprotein and LD lipoprotein enter the oocytes by a receptor-mediated transport mechanism; (2) the receptors for VLD lipoprotein and LD lipoprotein are distinct; and (3) the binding of LD lipoprotein to chicken oocyte membranes differs from that to other cell types.     

Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin.

Aspirin (acetylsalicylic acid) inhibits prostaglandin synthesis by acetylating an active site portion of the enzyme, prostaglandin synthetase. In the current study, the site of acetylation has been demonstrated to be a seryl residue at the NH2 terminus of the enzyme. Purified [3H]acetyl enzyme was prepared from seminal vesicle homogenates treated with [acetyl-3H]aspirin. The [3H]acetate to protein bond was stable to hydroxylamine, indicating an N-acetyl linkage. The [3H]acetyl enzyme was fragmented sequentially with cyanogen bromide, trypsin, and pronase. The 3H material isolated from the pronase digest was identified as N-acetylserine. This finding indicates that the oxygenase portion of prostaglandin synthetase has an NH2-terminal serine which is involved in enzymatic activity and is susceptible to acetylation by aspirin.     

Glutamic acid decarboxylase activity in striatal slices: Persistent increase following depolarization

When slices prepared from rat corpus striatum were preincubated for 15 min in potassium-enriched Krebs Ringer-Phosphate medium (K⁺-KRP), the activity of glutamic acid decarboxylase measured upon reincubation in normal Krebs-Ringer-Phosphate (KRP) was doubled as compared to GAD activity in slices preincubated in normal KRP. Similarly, when striatal slices were preincubated in KRP containing 100 μM veratridine, GAD activity upon reincubation in normal KRP was increased 66% as compared to activity in slices preincubated in normal KRP. The observed increase in GAD activity was not a function of alterations in glutamate uptake by the slices. These results suggest that GABAergic neurons may regulate transmitter synthesis during the process of depolarization by increasing GAD activity.