The addition of poly(ethylene glycol) (PEG) to biomolecules and biomaterials is a well-established approach to modify their properties for therapeutic applications. For biomaterials, the approach is typically to blend or electrospray the synthetic polymer with the biomaterial. Effective surface modification approaches such as surface-initiated polymer brushes are challenging since the harsh solvents required for brush synthesis may destroy the biomaterial. Herein, we describe the PEGylation of collagen fibers by surface-initiated PEG brushes using a living anionic grafting-from mechanism. This brush synthesis is done in the absence of solvents to minimize the degradation of the native collagen structure. We quantify the effect the brush synthesis has on the native structure of the collagen fiber using differential scanning calorimetry (DSC) and find that even at long reaction times a significant fraction of the native structure remains. Dynamic mechanical analysis indicates the collagen undergoes only modest structural degradation, while adhesion studies find a significant improvement of antifouling properties. Further, our approach opens the way for further chemistry, as the growing polymer chain is a potassium alkoxy group that can be functionalized by termination or by subsequent reaction by a wide variety of molecules.

1. The interaction at room temperature of thiols (cysteine and certain of its derivatives, and glutathione) with N-alkyl-N-nitrosourethanes and with diazomethane gave complicated mixtures of products. 2. S-Methylcysteine and S-ethoxycarbonylcysteine, the main products of the reaction between cysteine and N-methyl-N-nitrosourethane, were isolated and unequivocally identified. Paper-chromatographic and other evidence was used for the identification of several other components of the mixtures of products obtained. 3. S-Methyl derivatives and their methyl esters were the common products formed from the thiol compounds with both N-methyl-N-nitrosourethane and with diazomethane. 4. The esters were unstable and hydrolysed readily. 5. S-Ethoxycarbonyl derivatives, the primary products of the reaction of the thiols with N-alkyl-N-nitrosourethanes, isomerized to the respective N-ethoxycarbonyl derivatives when the pH increased above 7.0; the migration of the ethoxycarbonyl group from S to N was particularly easy with cysteine, probably owing to the spatial proximity of the amino group. 6. It is suggested that the non-enzymic reactions in vitro of thiols with both N-methyl-N-nitrosourethane and diazomethane may represent models for events in vivo and might help in the studies of the carcinogenic process.

Effects of methyl methanesulfonate (MMS) on mouse testicular cell kinetics and sperm chromatin structure were determined flow cytometrically. Mice were exposed to a single ip injection of saline containing 0 or 150 mg/kg MMS. Relative ratios of 1N, 2N and 4N testicular cells were not affected until 22 days postexposure. Ratios of 1N cell types were altered from 13 to 22 days and were near normal by 25 days. This study revealed an MMS induced alteration of chromatin structure in testicular, elongated spermatids by the sperm chromatin structure assay (SCSA), a flow cytometric measure of the susceptibility of acridine orange stained sperm DNA to denaturation in situ. The SCSA also detected alterations in cauda sperm chromatin structure at 3 days, which was 8 days prior to alterations in sperm head morphology, indicating the increased sensitivity of the SCSA. SCSA data were practically similar whether measuring either fresh or frozen/thawed sperm, or whether measured by two different types of flow cytometers: a) laser driven, orthogonal optical axis; or b) low cost mercury arc lamp system with epiillumination. The data support the model of Sega and Owens [Mutat Res 111:227-244:1983] that MMS alkylates cysteine-SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to broken chromosomes and mutations.

Misonidazole, metronidazole and nitrofurazone can enhance the cytotoxicity of melphalan in vitro if the cells are subjected to a hypoxic pretreatment with the drug prior to exposure of melphalan in air. Potentiation of melphalan is dependent upon the concentrations of the nitro compound and the duration of the hypoxic pretreatment. On a concentration basis, nitrofurazone was most effective at enhancing melphalan toxicity and metronidazole was the least effective. This potentiating activity correlates with the electron affinity of each drug.

To extend initial results on the antineoplastic activity of alpha-1,3,5-triglycidyl-s-triazinetrione (TGT, NSC 296934), a novel triepoxidic derivative, this compound was tested in a series of murine transplantable tumors. Repeated daily treatments with well-tolerated systemic doses of this chemical produced substantial retardation in tumor growth and significant prolongation of survival in the line 16 mammary, M5067 ovarian, and Madison 109 lung carcinomas and in mFS6 fibrosarcoma. Very marked activity was also seen in the P815 mastocytoma, B16 melanoma, line 38 colon carcinoma, and an intracerebrally transplanted ependymoblastoma, with high proportions of cures after one or two injections in IP transplanted SL2 lymphoma and line 26 colon carcinoma. It is concluded that the high level of antineoplastic effectiveness and the wide spectrum of TGT activity together with its novel structural characteristics could be of clinical significance.

Anti-(human lymphocyte) globulin was reacted with a mixed anhydride derivative of chlorambucil to give a product which was in turn reacted with diphtheria toxin. The resulting conjugate was partially purified and was found to possess an ability similar to that of the native antibody to bind to the human lymphoblastoid cell lines, CLA4 and Daudi. Daudi cells, as had been observed previously with CLA4 cells, lacked the high sensitivity to diphtheria toxin normally characteristic of cells of human origin. Thus treatment with free toxin at a concentration of 1 microgram/ml was without effect upon their ability to incorporate [3H]leucine. By contrast, Daudi cells were highly sensitive to toxin conjugated to anti-(human lymphocyte) globulin or to its F(ab')2 fragment. Exposure for 24 h to a solution of conjugate containing toxin at a concentration of 0.5 ng/ml caused a reduction of 50% in the leucine uptake by Daudi cells. The toxicity of the conjugate could be blocked by diphtheria antitoxin or by pretreatment of the cells with non-conjugated antibody. Toxin linked to normal horse IgG or to its F(ab')2 fragment was without cytotoxic effect upon Daudi cells. Furthermore both the conjugate with anti-(human lymphocyte) globulin and that with normal IgG were approximately 100-fold less able than non-conjugated toxin to inhibit protein synthesis by a human fibroblast cell line to which the antibody showed no appreciable binding. Thus the conjugates are relatively ineffective against cells which lack an antigen to which the antibody moiety can bind. In contrast with the greatly increased toxicity of diphtheria toxin for human lymphoblastoid cells following its linkage to anti-(human lymphocyte) globulin, a conjugate of toxin linked to anti-(mouse lymphocyte) globulin was ineffective against murine spleen cells in vitro.

Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% OsO4 buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice. If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [3H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10(5) deoxynucleotides, and gradually decreased to 2.2 ethylations/10(5) deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, and was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells. A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation of cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfide-bond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.