Poly(ethylene oxide)-grafted thermoplastic membranes for use as cellular hybrid bio-artificial organs in the central nervous system

Poly(acrylonitrile-co-vinyl chloride) (PAN/VC) anisotropic membranes were chemically modified with poly(ethylene oxide) (PEO) (5000 and 20,000 g/mol) by one of two aqueous reactions: (a) acid hydrolysis of the nitrile group to a carboxylic acid with which amine-terminated PEO (PEO-NH(2)) reacted or (b) base reduction of the nitrile group to an amine with which PEO-succinimide (PEO-SC) reacted. Approximately 1.3% of the bulk material was modified with PEO-NH(2) whereas 1.8 to 3.5% was modified with PEO-SC as determined by proton nuclear magnetic resonance ((1)H NMR) and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectra. Approximately 50 to 75% less bovine serum albumin (BSA) adsorbed to PEO-grafted single skin fibers than to unmodified PAN/VC. Transport properties of modified and unmodified fibers were compared by passive diffusion, convective nominal molecular weight cutoff, and hydraulic permeability. Neither hydraulic permeability nor nominal molecular weight cutoff of BSA changed appreciably after surface modification with PEO indicating that pore structure was not adversely affected by the chemistry involved in grafting poly(ethylene oxide). However, in the absence of any membrane conditioning, the apparent diffusion of alpha-chymotrypsinogen (24,000 g/mol) was enhanced in PEO-grafted PAN/VC fibers possibly as a result of reduced sorption of the permeating protein. In vivo biocompatibility in the brain tissue of rats was judged by histological assessment of the host's cellular response to fibers implanted for 30 days; biocompatibility of both PAN/VC and PAN/VC-g-PEO was satisfactory but improved slightly with PEO grafting. (c) 1994 John Wiley & Sons, Inc.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter--glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to wound signals in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.     

Mössbauer spectra of the heme peptide (HP) 1–50 and the heme peptide:non-heme peptide (NHP) non-covalent complex 1–50:51–104 derived from cytochrome c: evidence for cytochrome c iron site solvation in aqueous solution

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.     

Radiological evaluation of bone status in the jaw and in the vertebral column in a group of women

Vertebral bone mineral content was determined in a group of 56 women, ages 30–62. These measurements were compared with the status of supporting bone in the jaws (alveolar, molar and bicuspid) and with gingival health. There was a significant decline in vertebral bone mineral content from the pre- to post-menopausal group. Molar and bicuspid measurements were highly correlated. There was some association between lumbar bone mineral content and molar bone status for postmenopausal women. For postmenopausal women, the cases of greatest percent bone loss in alveolar crest were associated with lower lumbar bone mineral content. Gingival health did not confound the bone status measurements. The 56 subjects did not exhibit the degree of reduction in bone density that is observed in the general population. Further investigation using these radiographic techniques may reveal a link between substantial bone loss in the jaw and moderate to severe bone loss in the lumbar vertebrae.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.     

A first-generation map of the turkey genome.

A primary linkage map of the domestic turkey (Meleagris gallopavo) was developed by segregation analysis of genetic markers within a backcross family. This reference family includes 84 offspring from one F1 sire mated to two dams. Genomic DNA was digested using one of five restriction enzymes, and restriction fragment length polymorphisms were detected on Southern blots using probes prepared from 135 random clones isolated from a whole-embryo cDNA library. DNA sequence was subsequently determined for 114 of these cDNA clones. Sequence comparisons were done using BLAST searches of the GenBank database, and redundant sequences were eliminated. High similarity was found between 23% of the turkey sequences and mRNA sequences reported for the chicken. The current map, based on expressed genes, includes 138 loci, encompassing 113 loci arranged into 22 linkage groups and an additional 25 loci that remain unlinked. The average distance between linked markers is 6 cM and the longest linkage group (17 loci) measures 131 cM. The total map distance contained within linkage groups is 651 cM. The present map provides an important framework for future genome mapping in the turkey.     

Increased plasma corticosterone levels in bovine growth hormone (bGH) transgenic mice: Effects of ACTH,GH and IGF-I onin vitro adrenal corticosterone production

Previous work from our laboratory provided evidence for increased plasma corticosterone levels in mice transgenic for human and bovine growth hormone (GH). Corticosterone was elevated in both sexes, under both basal and ether-induced stress conditions. The objectives of the present study were to investigate thein vitro adrenal sensitivity to ACTH, GH and/or IGF-I in normal and bGH transgenic mice, to examine plasma corticosterone levels at different times of the day, and to determine plasma levels of ACTH in these animals. For the measurement of plasma corticosterone and ACTH levels, transgenic and normal siblings were housed 2 per cage and decapitated simultaneously within 20 seconds of the first disturbance of the cage. The corticosterone production byin vitro adrenal incubations did not differ between adrenals from normal and transgenic mice at the basal level or in the presence of different doses of ACTH. Growth hormone or IGF-I did not have any effect on corticosterone productionin vitro when given alone, and did not modify the effects of ACTH on the accumulation of corticosterone in the media. Plasma corticosterone concentrations were higher in transgenic than in normal animals in both morning and evening. Plasma concentrations of ACTH in animals killed in the morning were sharply increased in transgenic males as compared with their normal siblings. The results indicate that increased circulating levels of corticosterone in transgenic mice are not due to a potentiation of ACTH actions by GH or IGF-I, but rather to a chronic increase in plasma ACTH levels. The increase in ACTH is presumably a reflection of GH actions in the hypothalamic-pituitary system.     

Response regulators of bacterial signal transduction systems: Selective domain shuffling during evolution

Response regulators of bacterial sensory transduction systems generally consist of receiver module domains covalently linked to effector domains. The effector domains include DNA binding and/or catalytic units that are regulated by sensor kinase-catalyzed aspartyl phosphorylation within their receiver modules. Most receiver modules are associated with three distinct families of DNA binding domains, but some are associated with other types of DNA binding domains, with methylated chemotaxis protein (MCP) demethylases, or with sensor kinases. A few exist as independent entities which regulate their target systems by noncovalent interactions.In this study the molecular phylogenies of the receiver modules and effector domains of 49 fully sequenced response regulators and their homologues were determined. The three major, evolutionarily distinct, DNA binding domains found in response regulators were evaluated for their phylogenetic relatedness, and the phylogenetic trees obtained for these domains were compared with those for the receiver modules. Members of one family (family 1) of DNA binding domains are linked to large ATPase domains which usually function cooperatively in the activation of E. Coli ⁵⁴-dependent promoters or their equivalents in other bacteria. Members of a second family (family 2) always function in conjunction with the E. Coli ⁷⁰ or its equivalent in other bacteria. A third family of DNA binding domains (family 3) functions by an uncharacterized mechanism involving more than one a factor. These three domain families utilize distinct helix-turn-helix motifs for DNA binding.The phylogenetic tree of the receiver modules revealed three major and several minor clusters of these domains. The three major receiver module clusters (clusters 1, 2, and 3) generally function with the three major families of DNA binding domains (families 1, 2, and 3, respectively) to comprise three classes of response regulators (classes 1, 2, and 3), although several exceptions exist. The minor clusters of receiver modules were usually, but not always, associated with other types of effector domains. Finally, several receiver modules did not fit into a cluster. It was concluded that receiver modules usually diverged from common ancestral protein domains together with the corresponding effector domains, although domain shuffling, due to intragenic splicing and fusion, must have occurred during the evolution of some of these proteins.Multiple sequence alignments of the 49 receiver modules and their various types of effector domains, together with other homologous domains, allowed definition of regions of striking sequence similarity and degrees of conservation of specific residues. Sequence data were correlated with structure/function when such information was available. These studies should provide guides for extrapolation of results obtained with one response regulator to others as well as for the design of future structure/function analyses. Correspondence to: M.H. Saier, Jr.     

Habitat,diet, and activity patterns of free-ranging woolly spider monkeys (Brachyteles arachnoides E. Geoffroy 1806)

Results of a 10-month study of the ecology and behavior of free- ranging woolly spider monkeys (Brachyteles arachnoides)in Brazil show that these animals are strongly folivorous. Leaf-eating accounted for more than 50% of the total feeding time in all samples but one and accounted for more than 80% of the total feeding time in three samples. Mature foliage was routinely eaten. Woolly spider monkeys consistently spend more than 50% of each day quietly resting and sleeping. Animals travel little except when actively feeding and show low levels of social interaction. Such an activity profile suggests that woolly spider monkeys may often be living near the limits of their energetic resources. The social organization of the species is unusual for a folivorous primate in that small groups of females and associated immature animals confine their activities to discrete home-range areas, whereas males are itinerant, traveling over the home ranges of various female groups. Animals sharing a common home-range area show no permanent daily pattern of association other than that of mother-dependent offspring. Foraging alone or with few conspecifics should maximize each individual’s returns from foraging by minimizing the day range that must be traveled each day to locate foods while simultaneously lowering interference competition for higher-quality dietary resources.