Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Sugar and Alcohol Stabilization of Yeast in Sweet Wine

Secondary fermentation of sweet wine was prevented by the Delle stabilization procedure. For this procedure, advantage is taken of the inhibitory effects of high concentrations of sugar as well as of alcohol. Thus, relatively small amounts of wine spirits were added to fermenting musts to obtain stability, as compared to the conventional procedure in which larger amounts of alcohol are added and the inhibitory effect of alcohol only is considered. The Delle value is a function of the concentrations in the wine, after spirits addition, of alcohol and sugar. Delle values which gave stable wine were dependent on time of alcohol addition, on strain of wine yeast, and on composition of wine spirits. Fractional addition of spirits, concentration of SO(2), and clarity of must had little effect on the Delle value. Sensory comparison of wines especially prepared for tasting by the Delle procedure and by the conventional procedure showed the wines made by the Delle procedure to be superior in quality. Under proper storage conditions, the Delle wines were shown to be microbiologically stable and resistant to wine spoilage organisms.     

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.     

Helminths of rodents and marsupials from Papua New Guinea,with the description of two new species,Echinostoma echymiperae n. sp. (Digenea: Echinostomatidae) and Vampirolepis peroryctis n. sp. (Cestoda: Hymenolepididae)

The following rodents and marsupials from the Western Highlands of Papua New Guinea have been examined for helminths: Anisomys imitator, Melomys spp., Pogonomelomys ruemmleri, Rattus spp., Echymipera kalubu and Peroryctes raffrayanus. Two new species and a number of new host records are reported. Echinostoma echymiperae n. sp., a digenean from the intestine of Echymipera kalubu, is characterised by the number of collar spines, the body armature and the shape and position of the gonads. Vampirolepis peroryctis n. sp., a cestode from the intestine of Peroryctes raffrayanus, is characterised by the length of the rostellar hooks, the shape of the ovary, the arrangement of the testes in a triangle and the extent of the cirrus-sac. Hymenolepis aklei, H. bradleyi, H. antechini, H. bettongiae, H. cercarteti, H. isoodontis and H. potoroi are transferred to Vampirolepis as new combinations. E. kalubu is a new host for Linstowia semoni and Pogonomelomys ruemmleri is a new host for Hymenolepis diminuta. V. peroryctis is the first platyhelminth to be reported from Peroryctes raffrayanus and Raillietina (Raillietina) sp. the second to be reported from the genus Melomys.     

Host specificity ofAcanthoscelides quadridentatus andA. Puniceus [Col.: Bruchidae] for biological control ofMimosa pigra (with preliminary data on their biology)

The biology and host specificity of the seed-feeding bruchids,Acanthoscelides quadridentatus (Schaeffer) andA. puniceus Johnson, from Mexico were studied in quarantine facilities in Australia. Distinguishing characters in the genitalia of each species are illustrated. Oviposition was recorded on 16 of 73 species of plants tested but larvae died without entering pods or seeds, except onMimosa invisa K. F. P. von Martius andM. pigra L. Larvae entering seeds ofM. invisa died in the first instar. The bruchids were clearly specific toM. pigra and were subsequently released as part of a program for biological control of this weed in the Northern Territory, Australia in April 1983 and thailand in July 1984.      

Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm², respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts. © 1994 wiley-Liss, Inc.     

Estimating selection by comparing synonymous and substitutional changes

A higher ratio of substitutional to synonymous changes in between-species than in within-species comparisons has been taken as evidence for positive selection changing amino acids. A model is presented in which a difference of this kind arises as a result of purely neutral mutations, provided that the species compared are sufficiently different to approach a steady state between forward and backward mutation. In Neissseria, substitutions are twice as frequent, relative to synonymous changes, in between-species comparisons: it is shown that the data are consistent with the neutral model. The argument does not invalidate evidence for positive selection, for example in Drosophila, when the species compared are fairly similar.     

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H₂O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H₂O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H₂O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H₂O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc.