Alterations in cerebrospinal fluid uridine,hypoxanthine, and xanthine in head-injured patients

1. Examination of the cerebrospinal fluid (CSF) of head-injured patients reveals that the concentration of intraventricular xanthine is elevated and that of uridine is decreased relative to those of adult lumbar CSF. 2. No correlations were observed between CSF lactate and CSF hypoxanthine, xanthine, or uridine, suggesting that changes in purine metabolites and the pyrimidine nucleoside do not index similar cellular events as does lactic acid production. 3. Ventricular CSF from hydrocephalic infants had uridine and hypoxanthine concentrations not significantly different from those of normal adult lumbar CSF, but xanthine was significantly elevated. 4. Since uridine has anticonvulsant properties and is a crucial substrate for cerebral metabolism, it may be useful to evaluate this pyrimidine for use in the management of patients with head injury.     

Difference in teratogenic potency of ethylenethiourea in rats and mice: relative contribution of embryonic and maternal factors

Ethylenethiourea (ETU) is a potent teratogen in the rat but not in the mouse or any other species tested. Embryotoxic and teratogenic effects are produced in mice only after exposure to 10-40 times the teratogenic dose of ETU in rats. This study was undertaken to determine whether the difference in sensitivity between rats and mice is due to differences within the embryo, to maternal metabolic differences, or both. Comparably staged rat and mouse embryos (gestation day 10.5 and 8.5, respectively) with intact extra-embryonic membranes were maintained under identical conditions in whole embryo culture and exposed to static concentrations of ETU for 48 hours. The teratogenic effects of ETU were qualitatively similar in both species, characterized by excessive fluid accumulations in embryonic structures. The most common abnormalities were distended neural tube, especially in the hindbrain, and clear blisters on the caudal region. At least two times as much ETU was required to produce a similar incidence of abnormalities in mice as in rats. Thus, there is some intrinsic difference in the quantitative response of rat and mouse embryos to ETU, but it is insufficient to account for the in vivo discrepancy. The role of maternal metabolism in modifying the teratogenicity of ETU was assessed by adding hepatic S-9 fractions from Aroclor 1254-induced rats and mice to whole embryo culture. Rat S-9 had no effect on ETU teratogenicity. Mouse S-9 virtually eliminated the formation of abnormalities typical of ETU in both rat and mouse embryos. Decreased exocoelomic fluid osmolality, a physiological effect produced by ETU, also was not observed in embryos exposed to ETU and mouse S-9. ETU-typical defects were observed in embryos exposed to ETU and mouse S-9 which had been treated with carbon monoxide to inactivate its monooxygenase system, indicating that the mouse S-9 was metabolizing ETU. A surprising result was that adding mouse S-9 to embryo cultures containing ETU resulted in the formation of abnormalities (principally open neural tube) that were not observed in in vitro rat or mouse embryos exposed to ETU alone, or in mouse embryos in vivo. We believe that the most likely cause of these abnormalities is a putative ETU metabolite, which is rapidly excreted by the dam in vivo, but accumulates to teratogenic concentrations in vitro.     

Chemical evolution and the origin of life

This bibliography is the seventeenth annual supplement to the comprehensive bibliography on the same subject which was published in Space Life Sci. 2(1970), 225-295; 3(1972), 293-304; 4(1973), 309-329 and in Origins of Life 5(1974), 505-527; 6(1975), 285-300; 7(1976), 75-85; 8(1977), 59-66; 9(1978), 67-74; 10(1980), 69-87; 10(1980), 379-404; 11(1981), 273-288; 12(1982), 93-118; 13(1983), 61-80; 15(1984), 55-69; 17(1987), 171-184; 17(1987), 185-206.     

Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiation-induced cardiomyopathy in the dog

Sequential necropsies and histologic evaluations of young adult beagle dogs were performed after irradiation of the thorax. Total doses to the heart were 36, 44, or 52 Gy given in 4-Gy fractions in 4 weeks. One month after irradiation there was little histologic evidence of damage visible by light microscopy. However, ventricular and septal weights were increased, probably due to edema. At 3 months damage to endothelial and mesothelial cells was evident. By 12 months the myocardium was thinned and focal degeneration and loss of muscle cells and Purkinje fibers were observed. There was extensive subendocardial and epicardial fibrosis as well as intimal proliferation in coronary arteries. Morphometric analyses were performed on the myocardium, pericardium, atria, and aorta. There was a slight increase in perivascular connective tissue in the myocardium. The pericardium was increased in thickness and the ratio of smooth muscle to elastin was decreased in the aorta. Severe fibrosis occurred only in the right atrium. At 1 year there was no clinical evidence of heart failure; however, evidence of myocardial damage was present histologically and functionally. Additional stress and continued aging are likely to enhance the damage and lead to serious complications. The interactions of irradiated lung and heart require further investigation.     

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

In vivo formation and stability of engineered disulfide bonds in subtilisin

Computer modeling suggested that a disulfide bond could be built into Bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, Thr) and 87 (Ser) or between positions 24 (Ser) and 87 (Ser). Single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. The corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in Bacillus subtilis. Double-cysteine mutant enzymes were secreted as efficiently as wild-type, and disulfide bonds were formed quantitatively in vivo. These disulfide bonds were introduced approximately 24 A away from the catalytic site and had no detectable effect on either the specific activities or the pH optima of the mutant enzymes. The equilibrium constants for the reduction of the mutant disulfide bonds by dithiothreitol were determined to be 82 +/- 22 and 20 +/- 5 for Cys22/Cys87 and Cys24/Cys87, respectively. Studies of autoproteolytic inactivation of wild-type subtilisin support a relationship between autolytic stability and conformational stability of the protein. The stabilities of Cys24/Cys87 and wild-type enzymes to autolysis were essentially the same; however, Cys22/Cys87 was actually less stable to autolysis. Reduction of the disulfide cross-bridge lowered the autolytic stability of both double-cysteine mutants relative to their disulfide forms. This correlates with a lowered autolytic stability for the Cys22 and Cys87 single-cysteine mutants, and the fact that an intramolecular hydrogen bond between the hydroxyl groups of Thr22 and Ser87 is likely to be disrupted in the Cys22 and Cys87 single-cysteine mutant proteins.     

Movement of Fe with respect to the heme plane in the R-T transition of carp hemoglobin. An extended x-ray absorption fine structure study

Carp Hb undergoes a well known change in kinetics over the pH range 6-9. X-ray absorption spectroscopy, in conjunction with refined data analysis procedures, shows no difference in iron-ligand distances when carp HbCO is switched from R (high affinity) to T (low affinity) states. These distances are 2.015 +/- 0.015 A for the average iron-pyrrole nitrogen distance, 2.14 +/- 0.04 A for the iron-nitrogen (of histidine) distance, and 1.89 +/- 0.05 A for the Fe-C (of CO) distance. Examination of the region from 30 to 100 eV above the threshold, called the ligand field indicator region, reveals spectral differences, which when compared to model compounds suggest that the iron and the heme are less coplanar in the T-like forms. These results are consistent with the iron being 0.1 A more out of the mean heme plane in both carp HbCO and carp Hb T states, relative to the R forms, and that the change in iron position on ligation to either T or R state is four times larger than that occurring with the quaternary switch.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.