Role of subdermal current shunts in the failure of frogs to regenerate.

Large, uniform, skin-driven currents (20-40 muamp/cm2) leave the ends of limb stumps of post-metamorphic frogs (Rana pipiens) from about the first through the tenth day after amputation. However, right after amputation, while currents of comparable density may leave the periphery of the cut surface, current densities are greatly depressed in the center of this surface. We suggest that this depression is brought about by shunting through the subdermal lymph space (characteristic of anurans but not urodeles); continues in covert form after formation of a wound epithelium; and helps explain the ability of small, imposed currents to initiate frog limb regeneration.     

Biosynthesis of functionally active heparin cofactor II by a human hepatoma-derived cell line

Human plasma heparin cofactor II (HCII) inhibits thrombin by rapidly forming a stable, equimolar complex in the presence of heparin or dermatan sulfate. Cultured human hepatoma-derived cells (PLC/PRF-5) secreted (approximately equal to 200 ng/ml in 3 days) a protein of MW - 72 kD that was immunoisolated and immunoblotted with anti-HCII, co-migrated on SDS-PAGE with human plasma HCII, and formed covalent complexes with thrombin (MW - 101 kD) in the presence but not absence of heparin or dermatan sulfate; these complexes co-migrated with those obtained by incubating thrombin with human plasma under the same conditions. HCII was not detectable (less than 0.13 ng/ml) in post-culture medium from cultured human umbilical vein endothelial cells or human foreskin fibroblasts.     

Computer-Assisted Image Analysis of Plant Growth, Thigmomorphogenesis, and Gravitropism

A nonintrusive auxonometric system, based on the DARWIN image processor (Telewski et al. 1983 Plant Physiol 72: 177-181), is described and demonstrated in the analysis of gravitropism and thigmomorphogenesis in corn seedlings (Zea mays). Using this system, growth and bending of regularly shaped plants or organs can be quickly and accurately measured without, in any way, interfering with the plant. Furthermore, the growth and bending curves are automatically plotted. Thigmomorphogenesis in the aerial part of corn seedlings involves growth promotion at a low force load and growth retardation at higher force loads. The time courses of the two kinds of response are somewhat different, with retardation occurring immeditely after mechanical perturbation and growth promotion taking somewhat longer to begin. Gravitropic experiments show that when dark-grown corn seedlings are placed on their side in the light, the resulting curvature is due to two consecutive morphological mechanisms. In the first instance, lasting for about 15 minutes, the elongation of the bottom edge of the plant accelerates, while the elongation of the top edge remains constant. After that, for the next 1.75 hours, the elongation of the top edge decelerates and stops while that of the bottom edge remains constant at the increased rate for most of the period. The measurements taken from both experiments at relatively high resolution (0.08-0.1 millimeter) show that the growth curves are not smooth but show many small irregularities which may or may not involve micronutations.     

Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.

The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose dependent inhibition of B-16 melanoma growth in vivo by a synthetic analogue of PGE2

Daily intratumor administration of 16,16-dimethyl-PGE2-methyl ester in two different dosages inhibited tumor growth in C57Bl/6J mice bearing subcutaneous B-16 melanomas. The larger dose (20 microgram/day/mouse) produced a 68% decrease in tumor volume, a 69% decrease in tumor weight and a 60% decrease in the number of cells in mitotic phase. The smaller dose (10microgram/day/mouse) was one fifth less effective than the 20microgram dose but produced similar changes. Histological examination of tumors revealed no significant differences either in the inflammatory cell population or the amount of necrosis in the control and di-M-PGE2-treated tumors.     

Large electrical currents traverse growing pollen tubes

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.     

Epidemiological Characteristics of Strongyloidiasis in Inhabitants of Indigenous Communities in Borneo Island,Malaysia

Epidemiological study on strongyloidiasis in humans is currently lacking in Malaysia. Thus, a cross-sectional study was carried out to determine the prevalence of Strongyloides stercoralis infection among the inhabitants of longhouse indigenous communities in Sarawak. A single stool and blood sample were collected from each participant and subjected to microscopy, serological and molecular techniques. Five species of intestinal parasites were identified by stool microscopy. None of the stool samples were positive for S. stercoralis. However, 11% of 236 serum samples were seropositive for strongyloidiasis. Further confirmation using molecular technique on stool samples of the seropositive individuals successfully amplified 5 samples, suggesting current active infections. The prevalence was significantly higher in adult males and tended to increase with age. S. stercoralis should no longer be neglected in any intestinal parasitic survey. Combination of more than 1 diagnostic technique is necessary to increase the likelihood of estimating the ‘true’ prevalence of S. stercoralis.     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.