Mapping of antigenic sites to monoclonal antibodies on the primary structure of the F1-ATPase beta subunit from Escherichia coli: concealed amino-terminal region of the subunit in the F1.

To analyze relationships between the ternary and primary structures of the beta subunit of Escherichia coli F1 ATPase, we prepared two monoclonal antibodies beta 12 and beta 31 against the beta peptide. These antibodies bind to the beta subunit but do not bind to the F1 ATPase, resulting in no inhibition of the ATPase activities. Several different portions of the beta subunit peptide were prepared by constructing expression plasmids carrying the corresponding DNA segment of the beta subunit gene amplified by the polymerase chain reaction. Western blotting analysis using these peptides revealed that the antibodies bound to a peptide of 104 amino acid residues from the amino terminal end, which is outside the previously estimated catalytic domain between residues 140 and 350. These results indicated that the amino terminal portion of the maximal 104 residues is not exposed to the surface of the F1 ATPase. The binding spectrum of the antibodies to the subunit from various species including Vibrio alginolyticus and thermophilic bacterium PS3 indicated possible epitope sequences within the 104 residues. The ternary structure of the beta subunit, in terms of cleavage sites by endopeptidases, was analyzed using the antibodies. A 43-kDa peptide without binding ability to beta 12 and beta 31 appeared upon cleavage by lysyl endopeptidase. The results suggested that lysyl residues from around 70 to 100 from the amino terminus are exposed to the surface of the beta subunit.     

Synthesis of 4'-deoxy-4'-fluorokanamycin A and B.

4'-Deoxy-4'-fluorokanamycins A (17) and B (25) have been prepared through fluorinative ring-opening of the D-galacto-3',4'-oxiranes (8 and 21) derived from kanamycin A and B with potassium hydrogenfluoride in ethane-1,2-diol. The mechanism of preponderant formation of the 4'-deoxy-4'-fluoro-D-gluco (9 and 22) over the 3'-deoxy-3'-fluoro-D-gulo derivatives was discussed. In the synthesis of 25, the unusual 3',6'-epimine (23) was the main product along with the 4'-deoxy-4'-fluoro derivative. The mechanism of this reaction is also discussed. Both 17 and 25 were active against resistant bacteria producing aminoglycoside-adenylylating enzymes for HO-4'.     

Western ligand blot assay for human growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3): the serum levels in patients with classical growth hormone deficiency.

The insulin-like growth factors I and II (IGFs), important growth factors both in vivo and in vitro, are known to have at least six binding proteins (IGFBP-1-6). In human serum, IGFBP-3 is a major binding protein and is considered to be GH-IGF-I-dependent. We have established a Western Ligand Blot (WLB) assay for IGFBP-3. The method is a densitometric analysis of IGFBP-3 bands on a film of WLB. The IGFBP-3 levels of patients with classical growth hormone deficiency (GHD, 5 isolated and 10 multiple hormone deficiencies with appropriate therapy) were studied. Before puberty there is no overlap between control (n = 31) and the patients with GHD (n = 10). However, IGFBP-3 levels of two of five pubertal patients with GHD were within the normal range (n = 16). We think that measurement of serum IGFBP-3 is a useful diagnostic marker for GHD, especially before puberty.     

The Saccharomyces cerevisiae genes ( CMP1 and CMP2 ) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

Summary Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using¹²⁵I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.     

Absorption of cadmium after a long-term oral administration of cadmium to dogs

A long-term experiment using beagle dogs to investigate the absorption of cadmium was conducted. The dogs in the experimental groups were given a commercial diet and pelleted food containing 1, 3, 10, 50, and 100 mg of cadmium per day. The cadmium concentration in the blood increased continuously, gradually reaching a steady state following the administration of cadmium. The cadmium excreted daily in urine increased continuously. The cumulative excreted amount of cadmium in urine was calculated by using the trapezoidal rule based on the data of excretion of cadmium in urine. Then the absorbed fraction of administered cadmium was estimated on the basis of the relationship between the cumulative excreted amount of cadmium in urine and the cumulative administered dose of cadmium after the cadmium concentration in blood reached a steady state. The absorbed fraction of cadmium decreased with an increase in the administered dose of cadmium. A dose-dependent increase between the absorbed amount and the administered dose was observed.     

Family studies of the LDL receptor gene of relatively severe hereditary hypercholesterolemia associated with Achilles tendon xanthomas

Summary To assess the relationship between relatively severe hereditary hypercholesterolemia with Achilles tendon xanthomas and the defect of the low density lipoprotein (LDL) receptor gene, family studies were carried out in 17 hypercholesterolemic families. In 16 out of the 17 families, hypercholesterolemia co-segregated with four different gross rearrangements, six different restriction fragment length polymorphism (RFLP) haplotypes, or an abnormal TaqI band of the LDL receptor gene. These findings are compatible with the interpretation that hypercholesterolemia is caused by defective LDL receptor genes, and that the origin of the mutant LDL receptor genes in Japanese generally differs among different pedigrees. In the remaining family, the proband and his sibling, both having relatively severe hypercholesterolemia and Achilles tendon xanthomas, shared an RFLP haplotype, although the proband's other sibling with moderate hypercholesterolemia but without Achilles tendon xanthomas did not. The mutant gene for familial defective apolipoprotein B-100 was not detected in the 17 probands. These data suggest that most, if not all, of the relatively severe hereditary hypercholesterolemia associated with Achilles tendon xanthomas is caused by a defect of the LDL receptor gene.     

Apodemus sylvaticus, a new host for Acanthocheilonema viteae (Nematoda: filarioidea)

Susceptibility of Apodemus sylvaticus and A. agrarius to infection with Acanthocheilonema viteae was compared with that of hamsters and jirds. Microfilaremia in A. sylvaticus was first noted on day 52 post-infection (p.i.) and lasted during the course of the study (up to day 150 p.i.). Maximum microfilaremic levels (female worm basis) of A. sylvaticus [mean +/- S.D. (n) = 690 +/- 1288(6)] were considerably higher than those of hamsters [16 +/- 18(6)] and jirds [51 +/- 25(5)]. Adult worm recovery in A. sylvaticus ranged from 2 to 40% of the number of infective larvae inoculated. Worm development in A. sylvaticus resembled that in hamsters and jirds. In contrast, microfilaremia was not detected in, nor adult worms recovered from A. agrarius throughout the study.     

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.     

Escherichia coli mutants with altered cation recognition by the melibiose carrier.

Revertants that showed normal cation recognition for melibiose transport were isolated from mutants with altered cation recognition (W3133-2S and W3133-2T) of Escherichia coli. Although the original two mutants possessed a second alteration, an increased activity of the Na+(Li+)/H+ antiporter, the revertants, which possessed the normal melibiose carrier, still showed altered properties of the Na+(Li+)/H+ antiporter. These results support the view that the alterations in the melibiose carrier and in the Na+(Li+)/H+ antiporter, observed in the mutants, are not genetically linked.     

Inhibitory effect of Li+ on cell growth and pyruvate kinase activity of Escherichia coli.

Li+ inhibited growth of Escherichia coli when glucose, galactose, fructose, or glycerol was added as the sole source of carbon. Growth inhibition was not observed when lactate or a mixture of amino acids was used as the carbon source. A mutant possessing elevated activity of Li+ extrusion was not inhibited by Li+. These results suggested that intracellular Li+ inhibited the glycolytic pathway, most likely triose metabolism, without affecting gluconeogenesis. We also found that pyruvate kinase I was inhibited by Li+.