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Self-assembled monolayers (SAMs) of the aromatic diisocyanides, 1,4-phenylenediisocyanide, 2,3,5,6-tetramethyl-1,4-phenylenediisocyanide, 4,4‘-biphenyldiisocyanide, 3,3‘,5,5‘-tetramethyl-4,4‘-biphenyldiisocyanide, and 4,4‘ ‘-p-terphenyldiisocyanide, were prepared on gold and palladium surfaces. The SAMs were characterized by ellipsometry, polarization-modulated infrared reflection−absorption spectroscopy (PM-IRRAS), and grazing-angle attenuated total reflectance infrared spectroscopy (GATR). Based on the position of the metal-coordinated isocyanide stretching band, the SAMs on gold were found to bind in the terminal (η¹) geometry, while the SAMs on palladium prefer a different geometry which is possibly a triply bridging (μ3-η¹) geometry. A side-reaction of the unbound isocyanide in the SAM was identified as oxidation to an isocyanate group.

Modifications to DNA-5-methylcytosine (5MeC) content (i.e., alterations in the level of 5MeC) constitute epigenetic events. In general, hypomethylation of a gene is necessary but not sufficient for expression, while methylated genes typically are quiescent. Ha-ras is an oncogene commonly implicated in murine liver tumorigenesis, often, though not always, involving mutation. A PCR-based approach using pre-PCR digestion with methylation-sensitive enzymes was employed to determine the 5MeC content of the 5' flanking region of this gene in (i) B6C3F1 and C57BL/6 mouse liver from young animals (4 months old) and (ii) B6C3F1 mouse liver from aged animals (24 months old). Two segments of the 5' flanking region of Ha-ras were examined. We demonstrate the presence of 5MeC in a portion of the 5' flanking region of Ha-ras that does not share characteristics of a CpG island, while a region that shares CpG island characteristics is primarily unmethylated. Differences in methylation status in these areas of Ha-ras were not observed between B6C3F1 and C57BL/6 mouse livers. Increases in methylation status were observed with ageing in B6C3F1 mouse liver. These data provide a role for methylation in regulating Ha-ras expression in mouse liver. Ha-ras in human liver has been reported to be unmethylated. There are substantial sequence differences in a key region of the 5' flanking region of Ha-ras in mice as compared to humans. These differences in DNA methylation and sequence may, in part, provide a basis for the frequent involvement of Ha-ras in mouse liver tumors and its virtual lack of involvement in human tumors. Int. J. Cancer 72:491–497, 1997. © 1997 Wiley-Liss, Inc.

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.

Acute and subacute toxicology and safety evaluation was determined for hexahydro-1,3,5-triethyl-s-triazine (Vancide-TH), a bacteriostatic and fungistatic agent proposed for industrial use by R. T. Vanderbilt and Co., Inc. Single dose oral toxicity, subacute 14 day dermal toxicity and eye irritation studies were conducted on either the rat or the rabbit. The data obtained indicate that Vancide-TH should be classified as a "toxic, corrosive and an eye-irritating" substance as defined under the "Regulations" of the Federal Hazardous Substances Act. Since the acute dermal LD50 closely approximated the acute oral LD50 in rats, this route might constitute the most serious hazard involved with V-TH and special precautions should be exercised when handling or using this substance. Because of the severe eye-irritating action, precautions should be taken to protect the eyes when handling or using the compound; the ocular damage produced may be irreversible. The "in use" concentrations (1,000 to 10,000 ppm) produced a much lesser toxicity in animals and could be without hazard if precautions are taken.

The toxicological evaluation of a compound involves a variety of acute, subchronic, and chronic toxicity studies which over the past decade have greatly increased in size and complexity. The combination of voluminous amounts of data collected in chronic toxicology studies and regulatory requirements for extensive documentation of the toxicology testing process presents an enormous burden when manual processes are used for data collection, storage, and analysis. Toxicology studies using relatively standardized experimental protocols present an ideal opportunity for nearly complete automation and computerization of the data acquisition, storage, analysis, and reporting processes. The complexity of toxicology data collection and the requirement for flexibility to accommodate special needs for individual experiments necessitate, however, the use of sophisticated computer software and hardware on a scale that is often underestimated at the outset of a toxicology computer project by both toxicologists and computer specialists alike. This paper will provide an overview of the types of studies conducted during the toxicological evaluation of a compound, the types of data elements that are collected, and special requirements that are involved in the analysis and reporting of chronic toxicology/carcinogenicity and subchronic toxicity tests. In addition, an attempt will be made to provide a perspective on the magnitude of toxicology data processing involved in the safety evaluation process.

To explore further the structural requirements for ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of CYP2B induction by 5,5-diphenylbarbituric acid, phenytoin (5,5-diphenylhydantoin), barbital (5,5-diethylbarbituric acid) and 5,5-diethylhydantoin were investigated in the male F344/NCr rat. Steady-state total (free plus protein-bound) serum drug concentration, measured after 14 days of administration of the compounds in the diet, was used as an approximation of intrahepatocellular drug concentration. The serum concentrations associated with half-maximal hepatic CYP2B induction (EC50 values) were 6 to 11 microM and 15 to 18 microM for the diphenyl-substituted barbiturate and hydantoin, respectively, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values for the diethyl-substituted barbiturate and hydantoin were 16 to 20 microM and > or = 500 microM, respectively. The magnitudes of the maximal CYP2B induction responses elicited by the diphenyl-substituted congeners and by barbital were 94 to 122% of the responses resulting from phenobarbital itself. In contrast, the maximum induction responses elicited by 5,5-diethylhydantoin were only 24% as great as those elicited by phenobarbital. The finding of a CYP2B inducer with a potency value 2 to 3 orders of magnitude lower than those for certain other prototype CYP2B inducers is suggestive but not proof of receptor mediation in the induction process.