2,3-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol is a substrate for human glucocerebrosidase.

Glucocerebrosidase, the lysosomal enzyme that is deficient in patients with Gaucher's disease, hydrolyses non-physiological aryl beta-D-glucosides and glucocerebroside, its substrate in vivo. We document that 2,3,-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol (2,3,-di-14:0-beta-Glc-DAG) inhibits human placental glucocerebrosidase activity in vitro (Ki 0.18 mM), and the nature of inhibition is typical of a mixed-type pattern. Furthermore, 2,3-di-14:0-beta-Glc-DAG was shown to be an excellent substrate for the lysosomal beta-glucosidase (Km 0.15 mM; Vmax. 19.8 units/mg) when compared with the natural substrate glucocerebroside (Km 0.080 mM; Vmax. 10.4 units/mg). The observations that (i) glucocerebrosidase-catalysed hydrolysis of 2,3-di-14:0-beta-Glc-DAG is inhibited by conduritol B epoxide and glucosylsphingosine, and (ii) spleen and brain extracts from patients with Gaucher's disease are unable to hydrolyse 2,3-di-14:O-beta-Glc-DAG demonstrate that the same active site on the enzyme is responsible for catalysing the hydrolysis of 4-methylumbelliferyl beta-D-glucopyranoside, glucocerebroside and 2,3-di-14:O-beta-Glc-DAG. With the aid of computer modelling we have established that the oxygen atoms in 2,3-DAG-Glc at the C-1, C-4*, C-5* (the ring oxygen in glucose) and C-2 positions correspond topologically to the oxygens at C-1, C-4* and C-5* and the nitrogen atom attached to C-2 respectively in glucocerebroside (* signifies a carbon atom in glucose); furthermore, all of the distances with respect to overlap of corresponding heteroatoms range from 0.02 A to 0.77 A (0.002-0.077 nm). A root-mean-square deviation of 0.31 A (0.031 nm) was obtained when the energy-minimized structures of 2,3-di-14:O-beta-Glc-DAG and glucocerebroside were compared using the latter four heteroatom co-ordinates.

A case of nonneurologic Gaucher's disease that biochemically resembles the neurologic types.

Systemic findings such as hepatosplenomegaly and typical Gaucher storage cells in a bone marrow aspirate led to the clinical diagnosis of Gaucher's disease in the seven-year old patient described in this report. On the basis of the lack of neurologic involvement the child was classified as having the Type 1, nonneurologic form of Gaucher's disease. After splenectomy glucocerebrosidase was extracted from her spleen for biochemical analysis. As expected, a marked deficiency of glucocerebrosidase activity was evident in the splenic extract, however her enzyme displayed anomalous behavior compared to other identical splenic preparations from documented Type 1 Gaucher's disease patients in that it failed to reconstitute with the acidic lipid phosphatidylserine. Using the polymerase chain reaction (PCR)-based color complementation assay and restriction endonuclease analysis, we compared the mutation genotype of this child with that of five other classical Type 1 patients. This analysis revealed that our patient alone was homoallelic for a T----C transition at position 1448 in the glucocerebrosidase cDNA that results in a 444Leu----Pro substitution in the glucocerebrosidase protein. The latter mutation genotype is normally associated with the neurologic phenotype, namely, the Types 2 and 3 forms of the disease. The relevance of the nature of polarity in clinical and biochemical analyses is discussed with regard to the phenotypic classification and the future clinical course of disease in the child.

Analysis of paternity. The use of HLA and red cell antigens.

Over a 6-year period, 866 cases were analyzed for disputed parentage. In 194 cases (22%), the alleged father was excluded as the biological father. One hundred and eight-one cases (93%) were direct exclusions, and 13 cases (7%) were indirect exclusions. Ninety-two percent of falsely accused men were identified by typing for HLA antigens alone. Exclusions based solely on red cell data occurred in 8 percent of the cases. When only HLA testing was performed, 177 of 362 (43%) cases had a probability of paternity of greater than or equal to 95 percent. When both HLA and red cell typings were performed, 110 of 160 (69%) cases had a probability of paternity of greater than or equal to 95 percent. No significant differences in the proportion of exclusions or proportion of alleged fathers with a probability of paternity of greater than or equal to 95 percent were observed between Caucasians or blacks. We conclude that typing for HLA antigens provides the most powerful data for excluding or including an alleged father. However, typing for red cell antigens adds significant data in excluding a falsely accused man and in determining the probability of paternity at a critical level (greater than or equal to 95%). In addition, the current study suggests that additional assays are needed to identify genetic characteristics of cases with probability of paternity less than 95 percent.

Susceptibility of Staphylococcus aureus to cefaclor and cephalothin: laboratory and clinical studies.

In vitro susceptibility tests of 201 strains of Staphylococcus aureus by agar dilution revealed 90% to be susceptible to 8 mug or less of cefaclor per ml. Strains from hospitalized children and adults were more often resistant than those from patients with bullous impetigo. Cephalothin was more active than cefaclor against all strains tested. Results with disk tests, including those strains examined from the clinical investigation, revealed some discrepancies in identifying strains more resistant to cefaclor. In clinical studies, cefaclor proved quite effective for the treatment of bullous impetigo. Of 73 patients, 90% were cured and 7% improved after completing 5 or more days of treatment. Prompt improvement was noted among most patients seen 3 to 5 days after treatment was begun. One patient experienced mild diarrhea. There were no other adverse or toxic manifestations attributable to therapy.

Manner and meaning of susceptibility testing of ampicillin-resistant Haemophilus influenzae.

We examined ampicillin-resistant strains of Haemophilus influenzae to compare the percentage of resistant organisms in each strain with the susceptibility to ampicillin by an agar dilution method. Using an inoculum of 10(4) colony-forming units, the minimal inhibitory concentration (MIC) increased with the percentage of resistant organisms in the strain. Laboratory-manipulated strains composed of different proportions of a susceptible and a resistant strain behaved similarly. The survival of isolated colony-forming units (colony MIC) was then determined by spreading inocula over the surface of a set of MIC plates, resulting in separation of individual colonies. This modification of the susceptibility test to the colony level gave end points that were clear and reproducible and that did not vary with changes in incubation time or temperature. True differences in susceptibility among strains were demonstrated by this method, whereas results of the conventional MIC test may reflect only the number of resistant organisms present in the inoculum.