Gamma-T4 hybrid bacteriophage carrying the thymidine kinase gene of bacteriophage T4.

Among 32 lambda-T4 recombinant phages selected for growth on a thymidylate synthetase-deficient (thyA) host, 2 were shown to carry the T4 thymidine kinase (tk) gene. The lambda-T4tk phages contain two T4 HindIII DNA fragments (2.0 and 1.5 kilobases) that hybridize to restriction fragments of T4 DNA, encompassing the tk locus at 60 kilobases on the T4 map. The T4tk insert compensates for the simultaneous host deficiencies of thymidine kinase and thymidylate synthetase in a thymidine kinase-deficient (tdk) host growing in the presence of fluorodeoxyuridine when provided with thymidine and uridine. The lambda-T4tk hybrid phages specified five polypeptides with Mrs of 22,000 (22K), 21K, 14K, 11K, and 9K.     

Effects of cold stress and exercise on fat loss in females

A Latin Square design has been used to test the responses of 24 relatively fit young women to 200 minute bouts of exercise performed over 5 day trials under each of three different ambient conditions: 15 degrees C (warm-warm; (WW)); -20 degrees C while inhaling, from a facemask, air heated to 18 degrees C (cold-warm; (CW)); and -20 degrees C (cold-cold; (CC)). In both of the cold environments, special clothing and boots were provided (insulation 0.47 degree C X watt-1 X m-2 and 0.62 degree C X watt-1 X m-2; (4 and 3 CLO units)). All three trials led to a small (0.6-0.7 degree C) rise of rectal temperature, but in the two cold environments mean body temperatures fell by over 1.0 degree C. A large increase of serum ketones occurred under all conditions, and the exercise respiratory quotient suggested some increase of fat utilization, WW (0.85) through CW (0.84) to CC (0.83). A fat loss of about 0.5 kg over the five days was confirmed by hydrostatic weighing and measurement of skinfold thicknesses. This was much less than the change previously observed in men, and moreover, it seemed to be independent of ambient conditions. Possible reasons why cold did not increase fat loss in these women include: a lower relative intensity of exercise; a greater stability of fat stores in women; avoidance of caffeine; a possible translocation of subcutaneous fat to deep fat depots; and a greater desire to "lose weight" irrespective of environmental conditions.     

Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol. We have found that depolymerization of MTs by 1 microM Colcemid resulted in the rapid formation of massive IF-cables, structures distinct from "collapsed IFs" or "juxtanuclear coils." Neither the rapid formation of IF-cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 microM taxol rapidly formed MT-bundles, as well as aggregates of intertwining IFs, termed "IF-skeins." MT-bundles and IF-skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 microM taxol and 1 microM Colcemid were applied simultaneously, the complementary distributions of MT-bundles and IF-skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent. Decreasing the taxol: Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF-cables.     

Investigation of the attachment of bovine corneal endothelial cells to collagens and other components of the subendothelium : Role of fibronectin

Bovine corneal endothelial cells adhered equally well to a variety of collagens (types I, III, IV and V) consistent with a role for fibronectin in this process. They did not exhibit a preferential binding to collagen type IV—as might be anticipated if laminin were to play a significant role in their adhesion. Inhibition studies with anti-fibronectin antibodies demonstrated the importance of endogenous fibronectin in the mediation of attachment. Consistent with this, binding did not appear to require the presence of exogenous protein, since cells bound to collagens equally well in the presence or absence of added fibronectin and binding was not stimulated by pretreatment of collagens with this protein.     

Adenosine Triphosphatase Activity at the External Surface of Chicken Brain Synaptosomes

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.     

Pharmacokinetics of chlorogenic acid and rutin in larvae of Heliothis zea

Studies using [³H]chlorogenic acid and [³H]rutin demonstrated that the kinetics of uptake of these plant phenolics into the haemolymph of 5th-instar Heliothis zea (Boddie) following actue oral administration is a first-order process. The total quantity of either phenolic present in the haemolymph within 1 hr amounts to 5% or less of the total ingested dose. Based on TLC analyses, 80% or more of the radioactivity in the haemolymph occurs as the parent phenolic. Retention of [³H]-chlorogenic acid or [³H]-rutin in H. zea following chronic feeding from 1st to 3rd-instar larvae is also linearly related to dietary dose. Chlorogenic acid and rutin are both equitoxic and equivalent in bioavailability to H. zea.Loss of [³H]-rutin from the haemolymph of 5th-instar larvae following injection is biphasic. One half of the injected dose is excreted in the frass in the first 6 hr after injection; the other half is thereafter eliminated at 1/20th of the initial rate. Analyses of extracts of frass by thin-layer chromatography indicate that after either chronic or acute feeding 90% of the ingested phenolic is excreted unchanged. Possible sites and modes of action of phenolics in insects are discussed in light of these findings.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.