Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.     

Glyceryl monooleate as a fusogen for the formation of heterokaryons and interspecific hybrid cells

Glyceryl monooleate was used to induce heterokaryon formation between mouse LS fibroblasts and hen erythrocytes during 15 min incubation at 37 °C. Many of the heterokaryons that were formed contained haemoglobin since cell fusion occurred without complete haemolysis of the hen erythrocytes. Following fusion, the plasma membranes of the heterokaryons and their subcellular organelles were apparently intact and relatively undamaged. Although the results of individual experiments were variable, clones of viable hybrid cells were obtained on treatment of mouse 3T3 TK- fibroblasts and chinese hamster wg 3 IMP- fibroblasts with glyceryl monooleate up to 32 times more frequently than in untreated, control cultures. Karyogram analyses confirmed the presence of both sets of parental chromosomes in all hybrid cells studied. Clones of hybrid cells were isolated and subcultured successfully for several months in HAT medium.     

POLYMORPHISM IN THE DESMID XANTHIDIUM REGULARE AND ITS TAXONOMIC IMPLICATIONS

This paper refers to a case of polymorphism in the desmid genus Xanthidium Ehr. It is based on material from Lake Dais Irmaios, the main body of water in the Zoological and Botanical Garden in Recife, Pernambuco, northeastern Brazil, collected at 4 different times of the year during 1967 and 1968. A detailed examination of almost 1300 specimens showed an enormous variety in form of Xanthidium regulare Nordst., X. fragile Borge, and X. pseudoregulare Borge, thus allowing the authors to draw the following conclusions: (1) the name X. regulare Nordst. should be retained until further and more detailed studies on form variation within the species are available; (2) the names X. regulare Nordst. var. asteptum Nordst. in Borge, X. regulare Nordst. var. sexangulare Grönbl., X. regulare Nordst. var. sexangulare Grönbl. f. robustior Grönbl., X. fragile Borge, X. fragile Borge forma, and X. fragile Borge var. depauperatum Borge should be considered synonymous, all referring to a single variety of X. regulare Nordst., var. asteptum Nordst. in Borge emend. C. Bic. & L. M. Carv.; (3) X. pseudoregulare Borge must be treated as a variety of X. regulare Nordst. and must be called X. regulare Nordst. var. pseudoregulare (Barge) C. Bic. & L. M. Carv. Finally, a key is given to the 3 varieties of X. regulare Nordst. proposed in the present paper.     

Pathogenesis of hemosiderosis in lemurs: Role of dietary iron,tannin, and ascorbic acid

Clinical disease associated with excess iron deposition (hemosiderosis) in the duodenum, liver, and spleen occurs in captive lemurs. In this report we review the occurrence of hemosiderosis and related disease in the Zoological Society of San Diego lemur collection; we then define and describe potential pathogenic factors with the goal of establishing rational husbandry methods to limit or prevent the disease. At the San Diego Zoo, all 49 lemurs necropsied since 1968 were hemosiderotic, the severity increasing with increasing age; liver and kidney disease were common. Our review of iron metabolism, current knowledge on the pathogenesis of hemosiderosis in humans, and the diets of captive and wild lemurs reveals several key dietary substances that may contribute to lemur hemosiderosis: iron, tannins, and ascorbic acid. In captivity, excess dietary iron (commercial monkey chow) and high levels of ascorbic acid (citrus fruits) lead to enhanced iron uptake and increased toxicity of stored iron due to free radical formation. In the wild, lemurs have an unusual preference for leaves, fruits, and bark high in tannin, a polyphenolic secondary plant compound that rapidly chelates iron, protein, and minerals in the gastrointestinal (GI) tract, preventing their absorption. These findings suggest that hemosiderosis in captive lemurs results from a diet high in iron, high in ascorbic acid, and lacking in tannin. Immediate correction of captive diets may limit hemosiderosis in lemurs in the future.     

Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0.1-100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rare-cutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II variants have reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type II variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.     

Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies.

In an effort to determine the functional activity of anti-HIV-1 human mAb and to define the epitopes against which they are directed, supernatants from 10 EBV-transformed lymphoblastoid cell lines producing mAb to HIV were tested. Five clones producing mAb to gp41 and five producing mAb to p24 were identified. The anti-HIV-1 human mAb were tested in neutralization and cell fusion assays in the form of cell culture supernatants at concentrations ranging from 1.7 to 22.0 micrograms/ml. None of the human mAb were found either to inhibit HIV-1-(IIIB or RF) associated cell fusion or to neutralize HIV-1 (IIIB) infection of AA5 cells. All human mAb were additionally tested in 6 h 51Cr release assays for their ability to direct HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC). For ADCC assays, PBMC were isolated from healthy seronegative donors and used as effector cells. HIV-1 infected (IIIB, RF, and MN) CEM.NKR cells as well as CEM.NKR cells with purified gp120 adsorbed onto their surface served as targets. None of the anti-p24 mAb mediated ADCC. In contrast, three of the anti-gp41 mAb were able to direct a significant level of ADCC against each of the infected targets, but as expected, failed to lyse gp120 adsorbed cells. To define the specific epitopes against which the anti-gp41 mAb were directed, seven small peptides homologous to regions within the extracellular domain of gp41 were synthesized. Using RIA, two of the mAb could be mapped. The most effective ADCC-directing human mAb bound to a peptide comprising amino acids 644-663, whereas the least effective ADCC directing anti-gp41 human mAb bound to a region within the immunodominant portion of gp41 outlined by amino acids 579-604. Together, these results for the first time assign a functional activity to human mAb directed at specific regions within gp41 by demonstrating that areas within this molecule can serve as targets for ADCC.     

Contributions of left-handed helical residues to the structure and stability of bacteriophage T4 lysozyme

Non-glycine residues in proteins are rarely observed to have "left-handed helical" conformations. For glycine, however, this conformation is common. To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine. The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity. The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme. The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/mol). These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar. This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol. Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure. In the case of the Asn55 to Gly replacement, however, the (phi, psi) values of residue 55 change by about 20 degrees. This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues. This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (phi = 90 degrees, psi = 0 degrees), whereas that for non-glycine residues is close to (phi = 60 degrees, psi = 30 degrees). This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (phi, psi) energy contour maps.     

Elevated serine catabolism is associated with the heat shock response in Escherichia coli.

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.