Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Summary Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV 16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF-or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidinbiotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV 16 DNA and haptenized HSV2 DNA.     

Regional and cellular codistribution of interleukin 1 beta and nerve growth factor mRNA in the adult rat brain: possible relationship to the regulation of nerve growth factor synthesis

We have found a regional distribution of IL 1 beta mRNA and IL 1 activity in the normal adult rat brain, which reveals at least partially a colocalization with nerve growth factor (NGF). The predominantly neuronal signal patterns were found over the granule cells of the dentate gyrus, the pyramidal cells of the hippocampus, the granule cells of the cerebellum, the granule and periglomerular cells of the olfactory bulb, and over dispersed cells of the ventromedial hypothalamus and of the frontal cortex. In these areas also the highest levels of IL 1 activity were observed. In the striatum and septum much lower levels of IL 1 beta mRNA and IL 1 activity (shown for the striatum), most likely synthesized by glial cells, could be determined. IL 1 beta-expressing cells were mainly found in brain regions that also synthesize NGF mRNA as shown by in situ hybridization. NGF mRNA could be demonstrated over pyramidal cells of the hippocampus, granule cells of the dentate gyrus, periglomerular cells of the olfactory bulb and over prefrontal cortex neurons. These data indicate that IL 1 beta, among other factors, might also play a regulatory role in the synthesis of NGF in the CNS, as has been demonstrated in the peripheral nervous system (Lindholm, D., R. Heumann, M. Meyer, and H. Thoenen. 1987. Nature (Lond.). 330:658-659).     

Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately

The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins.     

Extraction of melodies in behavioural sequences

A method is proposed that extracts a set of phrases, or “melodies”, from a behavioural sequence, using a technique for extracting and compressing chains based on Information Theory. These melodies are validated by reference to a statistical criterion. An application of this method to the analysis of the behavioural sequences of two groups of mice, the first observed during the day, the second during the night, is described. The advantages and the limitations of the method are discussed.     

Different long-term effects of bilateral and unilateral nucleus basalis lesions on rat cerebral cortical neurotransmitter content

Young adult rats received either unilateral or bilateral ibotenic acid infusions in their nucleus basalis, destroying most of the cholinesterase-staining neurons in that region. Cerebral cortex levels of choline acetyltransferase, somatostatin, neuropeptide Y, and monoamines were then assayed 2.5 and 10 months after bilateral lesions, or, 2.5, 10, and 14 months after unilateral lesions. Entorhinal and cerebral cortex levels of several amino acid transmitters were also measured. As expected, choline acetyltransferase activity was decreased in the frontal cortex ipsilateral to the ibotenic acid infusion in unilaterally or bilaterally lesioned animals. Parietal cortex concentrations of somatostatin and neuropeptide Y were altered by lesioning in a complicated, time-dependent manner. Thus, while unilateral lesions transiently decreased or had no effect on these neuropeptide levels, bilateral lesions elevated the level of each neuropeptide by over 100% at 10 months. Other cortical transmitter systems investigated appeared to be less affected by nucleus basalis-lesions. Unilateral lesions had no effect on prefrontal cortex norepinephrine, serotonin, or dopamine content at 14 months post-lesioning. These different neurochemical effects of unilateral and bilateral nucleus basalis lesions may be important for developing a model for the trans-synaptic effects of cortical cholinergic deafferentation.     

Histochemical study on the eccrine glands in the foot pad of the cat

Enzyme and carbohydrate histochemical methods were used to study the secretory activities and secretion properties of the eccrine tubular glands in the foot pad of the cat. The activity spectra of the different oxidative and hydrolytic enzymes investigated indicate high metabolic rates within the secretory epithelium. Additionally the enzyme reactions emphasize a double innervation of the glands by cholinergic and adrenergic nerve fibres. The carbohydrate histochemical differentiation reveals mostly neutral and very few acidic glycoproteins in the secretory cells and the secretion, respectively. Clear (basal) cells contain distinct amounts of glycogen, and dark (superficial) cells show neutral glycoproteins, which reveal after PO-lectin-DAB procedures the following saccharide residues: alpha-D-mannose, alpha-D-galactose, N-acetyl-alpha-D-glucosamine, alpha-L-fucose, beta-D-galactose-D-N-galactosamine, beta-D-galactose, and sialic acid. The results obtained confirm the view that the normal biological functions of the eccrine glands of the feline foot pad are to improve the frictional capacities of the paw and to leave typical scent marks.     

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.     

Comparative measurements of the xylem pressure ofNicotiana plants by means of the pressure bomb and pressure probe

Determination of the pressure in the water-conducting vessels of intactNicotiana rustica L. plants showed that the pressure probe technique gave less-negative values than the Scholander-bomb method. Even though absolute values of the order of −0.1 MPa could be directly recorded in the xylem by means of the pressure probe, pressures between zero and atmospheric were also frequently found. The data obtained by the pressure probe for excised leaves showed that the Scholander bomb apparently did not read the actual tension in the xylem vessles ofNicotiana plants. The possibility that the pressure probe gave false readings was excluded by several experimental controls. In addition, cavitation and leaks either during the insertion of the microcapillary of the pressure probe, or else during the measurements were easily recognized when they occurred because of the sudden increase of the absolute xylem tension to that of water vapour or to atmospheric, respectively. Tension values of the same order could also be measured by means of the pressure probe in the xylem vessels of pieces of stem cut from leaves and roots under water and clamped at both ends. The magnitude of the absolute tension depended on the osmolarity of the bathing solution which was adjusted by addition of appropriate concentrations of polyethylene glycol. Partial and uniform pressurisation of plant tissues or organs, or of entire plants (by means of the Scholander bomb or of a hyperbaric chamber, respectively) and simultaneous recording of the xylem tension using the pressure probe showed that a 1∶1 response in xylem pressure only occurred under a few circumstances. A 1∶1 response required that the xylem vessels were in direct contact with an external water reservoir and/or that the tissue was (pre-)infiltrated with water. Corresponding pressure-probe measurements in isolated vascular bundles ofPlantago major L. orP. lanceolata L. plants attached to a Hepp-type osmometer indicated that the magnitude of the tension in the xylem vessels was determined by the external osmotic pressure of the reservoir. These and other experiments, as well as analysis of the data using classical thermodynamics, indicated that the turgor and the internal osmotic pressure of the accessory cells along the xylem vessels play an important role in the maintenance of a constant xylem tension. This conclusion is consistent with the cohesion theory. In agreement with the literature (P.E. Weatherley, 1976, Philos. Trans. R. Soc. London Ser. B23, 435–444; 1982, Encyclopedia of plant physiology, vol. 12B, 79-109), it was found that the tension in the xylem of intact plants under normal and elevated ambient pressure (as measured with the pressure probe) under quasi-stationary conditions was independent of the transpiration rate over a large range, indicating that the conductance of the flow path must be flow-dependent.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H(2)-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H(2)-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 +/- 0.19 mol%), a phospholipid pattern of type II, MK-9(H(4)) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t(d) = 8 h), H(2) plus CO(2) (t(d) = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H(2), nickel was not necessary. The optimum growth temperature was 65 degrees C; no growth was observed below 40 degrees C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70 degrees C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K(m) was 88 mul of CO liter and V(max) was 20.2 mul of CO h mg of protein. The threshold value of S. thermoautotrophicus of 0.2 mul of CO liter was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 mumol min mg of protein. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.