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Macrophage activation following attachment to biomedical polymers was studied using two systems of analysis. Supernatants generated by human peripheral blood monocytes cultured on the surface of several different biomedical polymers were evaluated for the presence of the secreted regulatory protein interleukin 1 (IL1). In addition, each cell-polymer culture surface was subjected to scanning electron microscopy for gross morphological evaluation. Results indicate that, although all materials were efficient in the attachment and activation of cells, the panel of polymers showed a differential capacity in attachment and activation of monocytes. Dacron and polyethylene surfaces had a greater density of cells showing morphology indicative of activation, corresponding to elevated levels of IL1 in these cultures. Biomer and polydimethylsiloxane surfaces showed fewer activated cells and had lower IL1 levels in culture. Expanded polytetrafluorethylene resulted in intermediate levels of IL1 and attached cells showing activated morphology.

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In vitro human platelet interactions with surfaces of type I collagen, chondroitin-6-sulphate (CH-6-S), chondroitin-4-sulphate (CH-4-S), a CH-6-S/collagen layer, and a collagen-CH-6-S complex were investigated. Polystyrene and silanized glass served as controls. Platelet counts, platelet factor 4 released, and platelet aggregating ability for the different surfaces were compared with controls. Platelet count and platelet factor 4 release data showed that there were no differences between surfaces of CH-6-S, CH-4-S and the controls. However, significant differences in platelet counts and platelet factor 4 released were found when collagen, the CH-6-S/collagen layer, and the collagen-CH-6-S complex were compared with controls. The pure type I collagen surface had the greatest influence on platelet activation. The collagen-CH-6-S complex had a greater effect that the CH-6-S/collagen layer on platelet activation. It appears that chondroitin-6-sulphate can modify the platelet activity of type I collagen.

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Mutation at the regA locus confers on somatic cells of Volvox (which otherwise undergo programmed death) ability to redifferentiate as reproductive cells. Stable mutations at the regA locus, but not at other loci, were induced at high frequency when embryos at one particular stage were exposed to either UV irradiation, novobiocin, nalidixic acid, bleomycin, 4-hydroxyaminoquinoline-1-oxide, 5-bromodeoxyuridine, or 5-fluorouracil. All treatments led to some mutations that were not expressed until the second generation after treatment. The sensitive period was after somatic and reproductive cells of the next generation had been set apart, but before they had undergone cytodifferentiation. Hypermutability occurs in presumptive reproductive cells (in which regA is normally not expressed) somewhat before regA normally acts in somatic cells. We postulate that hypermutability of regA in the reproductive cells at this time reflects a change of state that the locus undergoes as it is inactivated.

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The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m2) and subjected to variable length periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m2) is a nonlinear increase in cytotoxicity and is disproportionately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.

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Heparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

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A preliminary genetic analysis of a number of genetic variants of Volvox carteri f. nagariensis is presented. Techniques are outlined for mutagenesis of Volvox, isolation of mutants and routine genetic analysis. All of the mutants show simple Mendelian segregation patterns and have been tentatively placed in 14 linkage groups.

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High mol. wt. DNA was extracted from Escherichia coli lambda lysogens and was shown to be infectious. Its infectivity was due to prophage DNA integrated into the host chromosome rather than to DNA released from mature phage particles, as established by the following criteria: the titre of infectious DNA exceeded by 100-fold the titre of infectious units present before DNA extraction; mild shear selectively reduced prophage DNA infectivity to 2% of the unsheared DNA while lambda phage DNA infectivity retained 50% of its infectivity; DNA extracted from an E. coli (lambda c857 tsxisam6) lysogen yielded 200 times as many plaques on sup+ than on sup- spheroplasts. Thus lambda prophage DNA infectivity depends on expression of the excision gene while the infectivity of non-integrated forms of lambda does not. About 10(4) genome equivalents of E. coli DNA yielded one infectious centre unit in this assay system; this high infectivity should make prophage DNA a useful marker in genetic transformation experiments.

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Conditional lethal mutant systems developed in T-even bacteriophages T2, T4 and T6 have been used to study the partial exclusion which characterizes mixed infections of these phages. In bacteria mixedly infected with T2 and T4, the dominant phage (T4) acts against localized exclusion sensitivity determinants in the genome of the excluded phage (T2). These determinants are clustered near genes controlling early functions; the determinants themselves do not appear among the progeny, but markers located close to them appear infrequently, by recombination. The excluding action of T4 does not depend on the action of any gene so far identified by conditional lethal mutations, nor does it depend on differences in DNA glucosylation between infecting phages. Regardless of mechanism, the genetic consequence of this partial exclusion is to limit genetic exchange between T2 and T4 in the region of the genome controlling early functions, while retaining the capacity for extensive exchange in other regions; in short, partial exclusion constitutes a localized genetic isolating mechanism. Related forms of partial exclusion characterize mixed infections of other T-even phages, including those of some phages newly isolated from nature.

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Morphogenetic mutants of the colonial green alga, Volvox carteri f. nagariensis, were induced by chemical mutagenesis. The 68 independent mutants are classified into 12 readily identifiable phenotypes affecting various stages of asexual development. Nine of the mutants are temperature sensitive with normal development at 25 degrees and mutant development occurring at 35 degrees . Some mutant genes appear to be involved in the regulation of differentiation or the stability of the differentiated state. Other mutations occur in genes apparently responsible for structural components of dividing cells or adult colonies. Two mutants affect different aspects of the posterior-anterior polarization of the mature colony. One mutation affects a gene which acts very early in colonial development, but affects the appearance of the mature colony. The mutants isolated demonstrate the feasibility of using Volvox to study the genetic control of early steps in embryogenesis.

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The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.

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Multiplicity reactivation of bacteriophage inactivated by ultraviolet light is dependent on the recombination function of either the host bacterial cell or the infecting bacteriophage. Absence of both recombination systems leads to a loss of multiplicity reactivation.

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