The 165-kDa DNA topoisomerase I from Xenopus laevis oocytes is a tissue-specific variant.

Two forms of topoisomerase I can be purified from Xenopus laevis. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264, 4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a 110-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblotting or immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X. laevis tissue culture cells lysed and processed in the presence of SDS contain the 110-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Use of stream order and biological indices to assess water quality in the Osage and Black river basins of Missouri

Significant variations in four biological measures of water quality with stream order and river basin were demonstrated for streams of the Black and Osage river basins of Missouri. Water quality criteria specific for each order and basin were then developed.Benthic macroinvertebrates from springs and stream orders 3–8 in the two river basins were sampled quarterly for one year with riffle nets and artificial substrate samplers. A total of 548 samples were taken at 137 stations. The average annual macroinvertebrate density, index of diversity, number of taxa, and number of mayfly and stonefly taxa were determined for each station. These measures showed significant differences (p < 0.05) across stream order within and between the two river basins. Total taxa, total mayfly and stonefly taxa, and diversity were highest in orders 4 and 5 with decreased values in lower and higher stream orders. Maximum organism densities occurred in intermediate order streams. These differences were attributed to the succession of physical changes from headwaters to mouth within each river and to the unique geomorphology of each catchment basin.Water quality criteria based on three of the four measures described above (with 95% confidence limits) were established for each stream order in each river basin. Criteria for the Osage River basin were then used to identify three streams in the basin affected by environmental disturbances (stream impoundment, channelization and sewage discharge). The use of order- and basin-specific criteria assures that the biological differences between streams caused by environmental disturbance can be distinguished from the natural biological differences between streams of different orders and drainages.     

Chemoattraction of a bactivorous ciliate to bacteria surface compounds

The locomotory response to cell surface compounds extracted from two prey species,Vibrio natriegens andVibrio neries, was tested for a bacterivorous ciliate,Pseudochnilembus marinus Thompson 1966. Chemoattraction of the ciliate to the surface compounds stabilized in agarose baits was not equal for the two prey species. Fractionation of the extracts suggested the attractive substance was a high molecular weight compound. The expression of the differential response was dependant on the physiological condition and prior prey species exposure of the ciliate test population. The recognition and response to material normally found on the surface of prey cells supports evidence for the involvement of chemical sensing of gradients of prey particles and dissolved compounds of prey origin in the natural swimming behavior of bacterivorous ciliates. The prey species-specific reactions and influence of ciliate physiological state on chemosensory response suggest ciliate-bacteria interactions may be more complex than preciously assumed.     

Macrobenthic community structure before and after pollution abatement in the Neches River estuary (Texas)

Macrobenthos and physicochemical conditions in the lower 39 km of the Neches River estuary were studied from August, 1984 to May, 1985. The results were compared with data collected in 1971–1972. Between 1972 and 1984 the permitted BOD waste load in the tidal Neches River was reduced from 123 125 kg d to 8717 kg d. River discharge and dissolved oxygen concentrations were consistently higher and salinity was lower, during the same seasons, during the 1984–1985 study. A total of 50 taxa of macrobenthos were collected in 1971–1972 and 104 taxa were collected in 1984–1985. The numbers of taxa per collection at each station in 1984–1985 were at least twice those found in 1971–1972. Minimum densities in 1984–1985 were much higher than the maximum densities in 1971–1972 at all stations. Patterns of dominance, Sorenson's similarity index, and diversity ( ) values indicated improved water quality in 1984–1985. Statistical analysis of macrobenthic diversity indicated significant differences between upper estuary and lower estuary stations in 1971–1972. No significant differences were found in 1984–1985. Significant differences in numbers of taxa, macrobenthos densities, and values between the two studies were found. Reductions of waste loads, increased river discharge, and deepening of the navigation channel were among the factors that probably contributed most to the changes in community structure of the macrobenthos observed.     

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK _m over 120-fold but only decreases theV _max fourfold. SuchK _m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV _max 80-fold without changing theK _m .V _max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK _m andV _max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.     

Increase in β-1,4-Galactosyltransferase Activity During PC12 Cell Differentiation Induced by Forskolin and 2-Chloroadenosine

Galactosyltransferase (GALTase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chloroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine PC carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)