Solubility and diffusion coefficient of adenosine 3':5'-monophosphate.

Several previously unavailable parameters of adenosine 3':5'-monophosphate have been determined. The molar extinction coefficient at pH 7.0 is 1.38 X 10(-4), the aqueous solubility at pH 7.0 is 0.0236 M and the diffusion coefficient is 4.44 X 10(-6) cm2/s.     

Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.     

Eimeria tenella and E. acervulina: lymphokines secreted by an avian T cell lymphoma or by sporozoite-stimulated immune T lymphocytes protect chickens against avian coccidiosis

The role of avian lymphokines as nonspecific immunomodulators of host immunity against the intracellular parasite Eimeria was investigated. Prophylactic treatment of normal chickens with crude cell-free supernatants obtained from JMV-1 culture, concanavalin A (Con A)-stimulated normal spleen cells, or sporozoite-stimulated immune T cells prior to inoculation with E. tenella or E. acervulina conferred significant protection. These crude cell-free culture supernatants also inhibited intracellular development of eimerian parasites in vitro. Avian macrophages pretreated with these supernatant preparations showed inhibitory activity against Eimeria. This inhibitory activity could not be ascribed to anti-Eimeria antibody, complement, or cell-free Marek's disease virus and was therefore considered to be due to immunomodulating lymphokines present in the culture supernatants. These results suggest that JMV-1-transformed T lymphoblastoid cells, immune T lymphocytes, and Con A-stimulated normal spleen cells secrete lymphokines that can enhance host immunity in a nonspecific manner and implicate cell-mediated immunity as a major mechanism of the protective host immune response against eimerian infections.     

Modulation of cell surface heparan sulfate structure by growth of cells in the presence of chlorate

Swiss mouse 3T3 cells, when grown in the presence of 5 mM chlorate, an inhibitor of PAPS synthesis, produce heparan sulfate glycosaminoglycan chains containing only about 8% of the sulfate normally present and which have lost the ability to bind to fibronectin. These undersulfated chains are sensitive to nitrous acid at pH 4.5, indicating that many glucosaminyl residues have unsubstituted amino groups. The iduronic acid content of the heparan sulfate produced in the presence of chlorate is reduced to less than 7% as compared to the 36% in that from untreated cells. The chlorate-treated cells do not demonstrate any alterations in their growth control. However, the spreading behavior of these cells is altered to a flat rounded morphology compared to the more typical fibroblastic appearance of the untreated cell. The sulfation of chondroitin chains is also inhibited, but at a lower chlorate concentration which does not alter growth control or the spreading ability of the cells. These data indicate that (a) 3T3 cell surface heparan sulfate proteoglycan is not involved in growth control but may be involved in cell spreading, (b) the use of chlorate should be a valuable method for the study of the biosynthesis and structure/function relationships of sulfated glycosaminoglycans, and (c) the temporal sequence of the heparan sulfate chain modification reactions predicted from results of studies with cell-free extracts also operates in the cell.     

Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.     

A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation.

A heparin-binding protein was isolated from bovine uteri and purified to homogeneity. This protein appears as a double band of approx. 78 kDa in SDS/polyacrylamide-gel electrophoresis and has an isoelectric point of 5.2. The binding of heparin to this protein is saturable. No other glycosaminoglycan from mammalian tissue, such as hyaluronic acid, chondroitin sulphate, dermatan sulphate or keratan sulphate, binds to the 78 kDa protein. Dextran sulphate binds in a non-saturable fashion. Certain heparan sulphate polysaccharide structures are required for binding to the 78 kDa protein. Some proteoheparan sulphates, such as endothelial cell-surface proteoheparan sulphate, show only weak interaction with the 78 kDa protein in contrast with a basement-membrane proteoheparan sulphate from HR-9 cells. Antibodies against the 78 kDa protein inhibit binding of proteoheparan [35S]sulphate from basement membranes to smooth-muscle cells. Conventional antibodies, Fab fragments and some monoclonal antibodies, inhibit smooth-muscle cell proliferation in a similar range as that observed for heparin. The protein was detected in a variety of tissues and cells but not in blood cells. A possible role of this protein as a receptor for heparin or heparan sulphate and its function in the control of the arterial wall structure are discussed.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

Cancer: improving early detection and prevention. A community practice randomised trial.

OBJECTIVE--To test the impact of physician education and facilitator assisted office system interventions on cancer early detection and preventive services. DESIGN--A randomised trial of two interventions alone and in combination. SETTING AND SUBJECTS--Physicians in 98 ambulatory care practices in the United States. INTERVENTIONS--The education intervention consisted of a day long physician meeting directed at improving knowledge, attitudes, and skills relevant to cancer prevention and early detection. The office system intervention consisted of assistance from a project facilitator in establishing routines for providing needed services. These routines included division of responsibilities for providing services among physicians and their staff and the use of medical record flow sheets. MAIN OUTCOME MEASURES--The proportions of patients provided the cancer prevention and early detection services indicated annually according to the US National Cancer Institute. RESULTS--Based on cross sectional patient surveys, the office system intervention was associated with an increase in mammography, the recommendation to do breast self examination, clinical breast examination, faecal occult blood testing, advice to quit smoking, and the recommendation to decrease dietary fat. Education was associated only with an increase in mammography. Record review for a patient cohort confirmed cross sectional survey findings regarding the office system for mammography and faecal occult blood testing. CONCLUSION--Community practices assisted by a facilitator in the development and implementation of an office system can substantially improve provision of cancer early detection and preventive services.     

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

The fate of exogenous DNA introduced into Chlamydomonas reinhardtii by electroporation was analyzed. With single and double electrical pulses, plasmids as large as 14 kb were introduced into cells with and without intact cell walls. Within hours after introduction, exogenous plasmid DNA was associated with nuclei isolated from cells; several weeks after introduction, exogenous DNA was stably integrated into the Chlamydomonas genome. These studies establish electroporation as a method for introducing DNA, and potentially other molecules, into C. reinhardtii.