Detection of different cellular sides in rat liver and kidney by two monoclonal antibodies raised against the nucleotide-sugar hydrolyzing enzymes phosphodiesterase I and CMP-sialic acid hydrolase

Six different monoclonal antibodies were generated after immunization of mice with a partially purified enzyme preparation of rat liver, containing nucleotide-sugar hydrolase (NSH) I and II. These enzymes are also known under the names phosphodiesterase I and CMP-sialic acid hydrolase respectively [11]. In the enzyme-immunoassay the antibodies directed against NSH I displayed some cross-reactivity with the enzyme preparation of NSH II, and to a much lower extent the reverse was also true. Two antibodies, C and D highly reactive with NSH II and NSH I respectively, were used for immunocytochemical studies on sections of various rat tissues, which were known to contain high activities of both enzymes. Both antibodies were shown to be highly specific domain markers for different sides of the various cells. Antibody C was bound exclusively to the sinusoidal side of liver hepatocytes and to the basal side of cells from kidney tubule and epididymis. For antibody D the binding pattern was completely different, showing exclusive binding to the canalicular side of the hepatocytes and to the brush border membranes of kidney tubule cells, whereas in epididymis only binding to connective tissues was observed. Our studies clearly demonstrate, at least for liver and kidney, that NSH I and II are located at different cellular sides and that the monoclonal antibodies C and D can be used as domain markers for basal and apical sides of these cells respectively.     

A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom

The refined high resolution crystal structure of the bovine phospholipase A2 was compared with its counterpart from the venom of Crotalus atrox, the western diamondbacked rattlesnake. The strong similarity in their backbone conformations forms the basis of a common numbering system for the amino acid sequence. The three common major helices and much of the extended chain form a nearly identical "homologous core" structure. The variations in conformation usually arise from deletions/insertions or en bloc shifts of structural units. The exception to this is part of the highly conserved calcium-binding loop; however, this is to be expected as 1) there is no calcium ion sequestered in the venom dimer as there is in the case of the bovine enzyme and 2) two side chains in that segment form dimer-stabilizing interactions between the subunits of the C. atrox enzyme. The absolutely conserved catalytic network of hydrogen-bonded side chains formed by His 48, Tyr 52, Tyr 73, and Asp 99, as well as the hydrophobic wall that shields it, are virtually superimposable in the two structures. However, the details of the structural relationship between the amino terminus and the catalytic network differ in the two species and the ordered water molecules thought to be either functionally or structurally important in the pancreatic enzymes are not found in the crystal structure of the phospholipase A2 from C. atrox. The most striking difference from a functional standpoint is the fact that the surface depression in the region of the catalytic network that has been commonly considered the active site is shielded substantially in forming the intersubunit contact surface of the dimeric venom enzyme.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Double translocation t(7;12),t(2;6) heterozygosity in one family

Summary Double translocation heterozygosity t(2;6),t(7;12) in three generations of a Dutch family is described: the segregation of a double translocation in more than one generation has not been previously published. The index case was a 16-year-old mentally retarded boy with partial trisomy 12p who showed several dysmorphic features such as high prominent forehead, flat face, flat and short nose bridge, short nose, dysplastic ears, prominent lower lip, and several skeletal abnormalities. Based on the findings in this patient and those in nine other cases, the existence of a specific trisomy 12p syndrome is postulated.     

An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum

Acyl coenzyme A: isopenicillin N acyltransferase (AT) from Penicillium chrysogenum is an enzyme of interest for the biosynthesis of beta-lactam antibiotics. Severe aggregation problems with wild type AT have, however, prevented significant progress in the structure-function analysis of this enzyme for a decade. In this study, we show an approach to solve this aggregation problem by using dynamic light scattering (DLS) analysis to probe the aggregation state of the protein in the presence of various additives. After a one-step purification of recombinant wild type AT with a C-terminal His-tag using Ni2+ affinity chelate chromatography, addition of a combination of 5 mM DTT, 250 mM NaCl, and 5 mM EDTA to the purified AT effectively prevented aggregation. In the presence of these additives, the DLS profile of AT shows a narrow size distribution indicative of a homogeneous protein solution and the absence of aggregation. The purity and mono-dispersity of wild type AT was sufficient for the growth of high quality crystals diffracting to 1.64 A resolution.     

Effect of dietary energy source on energy balance, production, metabolic disorders and reproduction in lactating dairy cattle

The pathway for oxidation of energy involves a balanced oxidation of C2 and C3 compounds. During early lactation in dairy cattle this C2/C3 ratio is out of balance, due to a high availability of lipogenic (C2) products and a low availability of glycogenic (C3) products relative of the C2 and C3 products required for milk production. This review compares studies which manipulated dietary energy source and shows that dietary energy source can affect the balance of the C2/C3 ratio, as indicated by plasma NEFA, beta-hydroxybutyrate (BHBA) and glucose levels. It is shown that glycogenic nutrients increase glucose and insulin concentrations and decrease NEFA and BHBA plasma levels. Extra lipogenic nutrients elevate NEFA and BHBA and decrease plasma glucose concentrations. Lipogenic nutrients generally increase milk fat percentage and decrease milk protein percentage, suggesting a surplus of C2 compounds. The inverse is the case for feeding extra glycogenic nutrients, implying reduced deamination and oxidation of glycogenic amino acids. Feeding extra glycogenic nutrients improved the energy balance (EB), in contrast to ambiguous results of lipogenic nutrients on EB. Moreover, glycogenic feed may reduce the severity of ketosis and fatty liver, but increased the incidence of (sub)clinical acidosis. Since studies are scarce, it seems difficult to draw conclusions on the effects of dietary energy source on reproduction. However, lipogenic nutrients decrease glucose and increase NEFA and BHBA plasma levels. High plasma NEFA and BHBA and low plasma glucose levels are associated with decreased reproductive performance, which might imply the C2/C3 compound balance to be important for reproductive function.     

Identification of distinct Campylobacter lari genogroups by amplified fragment length polymorphism and protein electrophoretic profiles

Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters (NARTC) and the biochemical C. lari variants, including the urease-positive campylobacters (UPTC), the nalidixic acid-susceptible campylobacters (NASC), and the urease-producing nalidixic acid-susceptible campylobacters. To study the taxonomic and epidemiological relationships among strains of the C. lari variants, amplified fragment length polymorphism (AFLP) profiling and whole-cell protein profile analysis were performed with 55 C. lari strains. Great genetic heterogeneity in AFLP and protein profiles was observed. Numerical analysis of AFLP profiles and of partial protein profiles allowed discrimination of four distinct genogroups. AFLP cluster I included nearly homogeneous patterns for C. lari NARTC strains (genogroup I). UPTC strains together with non-urease-producing NASC strains produced highly diverse patterns and were placed in genogroup II. The genogroup III strains had the NASC phenotype and produced more homogeneous patterns. Finally, genogroup IV strains had the classical NARTC phenotype and produced AFLP patterns that were very distinct from those of other genogroups. One UPTC strain had aberrant patterns and clustered separately, which may indicate that there is an additional genogroup. Preliminary DNA-DNA hybridization experiments suggested that genogroups I and III represent a single genomic species and that genogroup IV represents a distinct species. The detection of moderate levels of DNA-DNA hybridization between a genogroup II reference strain and genogroup I and III reference strains highlights the need for further DNA-DNA hybridization experiments to clarify the taxonomic status of the former group. No correlation of genogroups with different sources of strains was identified. These data show that UPTC strains are genetically diverse and distinct from NARTC strains. In addition, they indicate that the classical NARTC phenotype encompasses at least two genogroups.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.