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We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

Abstract Plant-parasitic nematodes are important and cosmopolitan pathogens of crops. Here, we describe the generation and analysis of 1928 expressed sequence tags (ESTs) of a splice-leader 1 (SL1) library from mixed life stages of the root-lesion nematode Pratylenchus penetrans. The ESTs were grouped into 420 clusters and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. Approximately 80% of all translated clusters show homology to Caenorhabditis elegans proteins, and 37% of the C. elegans gene homologs had confirmed phenotypes as assessed by RNA interference tests. Use of an SL1-PCR approach, while ensuring the cloning of the 5′ ends of mRNAs, has demonstrated bias toward short transcripts. Putative nematode-specific and Pratylenchus -specific genes were identified, and their implications for nematode control strategies are discussed.

Concentrations of zinc and copper in a series of large oligotrophic lakes on Vancouver Island, Canada, have been increasing since mining operations began near the south end of Buttle lake in 1966. Declines in species diversity of both phytoplankton and periphyton have occurred since the beginning of mining activity with the disappearance of metal sensitive forms such asAsterionella andRhizosolenia. Cladoceran and calanoid copepod numbers and species diversity increase as the metal concentrations decline downstream from the mine. Bioassays carried out withDaphnia pulex show increasing toxicity downstream. Metal concentrations in Zooplankton and metallothionein concentrations in rainbow trout liver are correlated to metal concentrations in the water. The water was not acutely toxic to rainbow trout in laboratory simulations of Buttle Lake conditions or duringin situ exposures (1 month). Specific sublethal deleterious effects have not yet been determined.

Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.

Human lysosomal beta-galactosidase catalyzes the hydrolysis of beta-galactosides via a double displacement mechanism involving a covalent glycosyl enzyme intermediate. By use of the slow substrate 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, a glycosyl enzyme intermediate has been trapped on the enzyme. This has allowed the catalytic nucleophile to be identified as Glu-268 by peptic and tryptic digestion of the inactivated enzyme followed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry of the peptide mixture. This glutamic acid is fully conserved in a sequence-related family of enzymes (Family 35), consistent with its essential role.

2-Deoxy-2-fluoro-beta-glucosyl and -beta-mannosyl fluorides administered to rats in a single dose (10 mg/kg) inhibited beta-glucosidase and beta-mannosidase activity respectively after 1 h in brain, spleen, liver and kidney tissues. This inhibition, presumably caused by accumulation of 2-deoxy-2-fluoroglycosyl-enzyme intermediates, indicates that intact 2-deoxy-2-fluoroglycosyl fluorides are distributed to these organs and, in the case of brain, that they cross the blood/brain barrier. beta-Glucosidase activity recovered completely or partially in brain, spleen, liver and kidney by 20-48 h. beta-Mannosidase activity partially recovered in all tissues by 48 h. beta-Galactosidase activity in brain and kidney was not significantly affected by administration of either the gluco or manno compounds at this dosage, indicating that these inhibitors are directed towards specific glycosidases. Observation of similar relatively rapid rates of beta-glycosidase re-activation in vivo and in tissue homogenates in vitro at 37 degrees C suggests that hydrolysis or transglycosylation of 2-deoxy-2-fluoroglycosyl-enzymes, not protein synthesis, are the primary mechanisms involved in the recovery of glycosidase activity inhibited by this class of compounds in vivo.

Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2′,4′-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.

Synopsis This paper examines the relationship between mix proportions, electrical properties of the constituents of concrete and the over-all electrical resistivity for concrete. The mechanisms for the conduction of electricity through the heterogeneous medium of concrete are discussed and an electrical model is proposed. Analysis from a purely theoretical standpoint is also described and the values of electrical resistivity obtained experimentally are shown to compare favourably with the theoretical model.

Yeast alpha-glucosidase is a member of a sequence-related family of alpha-glycosidases (Family 13) that includes important digestive alpha-amylases and alpha-glucosidases. These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a glycosyl-enzyme intermediate. This intermediate can be trapped by use of 5-fluoro-alpha-D-glucosyl fluoride or 5-fluoro-beta-L-idosyl fluoride, members of a new class of mechanism-based glycosidase inactivators. Both of these trapped 5-fluoro glycosyl enzyme intermediates are catalytically competent, turning over when freed of excess inactivator and releasing free enzyme. Two glycosylated peptides in proteolytic digests of these trapped glycosyl enzyme intermediates were identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Further tandem mass spectrometric analysis in daughter ion scan mode allowed identification of Asp-214 as the catalytic nucleophile in yeast alpha-glucosidase, and this identification was confirmed by aminolysis of the labeled peptide and high resolution mass spectrometry. This residue is one of three active site carboxylates that are completely conserved in this family, thus confirming the role of Asp-214 and the equivalent residues in other family members as the catalytic nucleophile. The other two conserved carboxylates are likely involved in acid/base catalysis.

An assay for Moloney murine leukemia virus was developed that made use of the production of morphologically altered foci in nonproducer mouse cells (15F) carrying murine sarcoma virus. Wild-type (wt) virus gave a ratio of titers at 39 degrees C/34degrees C = 1.05 +/- 0.45 (standard deviation;n = 20). A spontaneous, thermosensitive (ts) mutant of Moloney murine leukemia virus, ts3, defective in a late viral function, gave 39 degrees C/34degrees C = 0. A murine cell line (TB) was mixedly infected with ts3 and wt (multiplicities of infection, 7.8:4.3), cloned after infection, and shown to be infected by both viruses. At 34 degrees C it produced wt, ts, and particles of mixed parentage. The heterozygotes (hz) had ratios of assays 39 degrees C/34 degrees C = 0.06 to 0.84 (mean, 0.36). To eliminate possible interference by multiploid particles with determination of the proportions of the three types of particles, the virus produced by the mixedly infected, cloned cell line at 34 degrees C was distributed by velocity sedimentation in a sucrose gradient, and virus was picked from the lightest part of the gradient. The proportions of ts, wt, and hz were 0.27, 0.26, and 0.47. Those particles identified as hz segreated ts, wt, and hz in the proportions 0.24, 0.27, and 0.49, respectively. These values were not significantly different from those predicted from a diploid model of the genome.

The BEST established feature of the neuropathology of poliomyelitis is the destruction of anterior horn cells of the spinal cord, this being generally recognized as the injury responsible for the enduring paralysis and atrophy of muscles of the trunk and extremities which may occur in this disease. Similarly, when present, paralysis of the cranial musculature or that of the oropharynx has been attributed to involvement of the analogous motor nuclei of the cranial nerves in the brain stem. As a consequence, and in spite of much evidence opposing such a view (Howe and Bodian1), there appears to be at least the popular impression that the only symptomatically important injury to the nervous system associated with poliomyelitis is the decrease in motor neuron collections.This concept was recently challenged by Kabat and Knapp,2 who called attention to the frequent involvement of interneurons in the gray matter of the spinal

THE origin of the murine sarcoma viruses (MuSV) found in tumours resulting from the injection of mice with leukaemo-genic viruses (MuLY)1–3and from spontaneous tumours3,4is very obscure. Viruses isolated from spontaneous tumours3,4might have been induced through accidental horizontal transmission of Moloney sarcoma virus. The origin of isolates of Moloney sarcoma virus1,2and Kirsten sarcoma virus5is also unknown because the virus preparations used had been passaged through rats2,5or mice of various strains for many years1. Nor is it known whether sarcoma virus was present in the preparations of murine leukaemia virus administered to the animals subsequently developing sarcomas. It has been suggested that Moloney MuSV is derived from Moloney MuLV6, but there may be an endogenous, latent sarcoma virus present in mice which could perhaps be activated by the administration of a leukaemogenic virus. We now report that a clone-purified MuLV, isolated from an established cell line in tissue culture and having in vitro no demonstrable content of MuSV, induced sarcomas in several strains of mice and MuSV could be readily isolated from these sarcomas. The virus was also present in the plasma of the tumour-bearing mice.

EARLY effects of carcinogenic hydrocarbons on the cell cycle and on deoxyribonucleic acid (DNA) synthesis have been noted by several authors. McCarter and Quastler1,2found that 7,12-dimethylbenz[α]anthracene (DMBA) decreases the rate of incorporation of 3H-thymidine into DNA and prolongs the DNA synthetic phase of the generation cycle of mouse epidermal cells. Evensen3found that 3-methylcholanthrene alters the proportion of epidermal cells labelled with 3H-thymidine in the hairless mouse. Jensen et al.4reported a reduction in uptake of 3H-thymidine in various tissues of the mouse following the administration of DMBA, including some tissues in which tumours do not usually arise after treatment with this agent. Elgjo5reported that 3-methylcholanthrene changes the mitotic phase of the cell cycle over an eleven-week period. We now report an early effect of DMBA on the synthesis of ribonucleic acid (RNA) in mouse skin.

The injection of 60 micrograms of 9,10-dimethyl-1,2-benzanthracene into newborn mice gave rise to a very high incidence of malignant thymomas. The tumor incidence was directly related to the dose of the carcinogen. The neonatal injection of the carcinogen also resulted in a depression in the immune response when the animals were challenged with an antigen as early as 4 weeks or as late as 11 weeks after administration of the carcinogen.

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THE carcinogenic hydrocarbon 7,12-dimethylbenz-[α]anthracene (DMBA) inhibits the incorporation of tritiated thymidine into the deoxyribonucleic acid (DNA) of the cells of the growing hair follicle of the mouse to the extent of approximately 50 per cent in 24 hr.1. We have now examined the effect of this hydrocarbon on the timing of the phases of the pro-liferative cycle of these cells.