The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid

A monoclonal antibody, 3F12, was generated which reacted specifically against infective or metacyclic stage Leishmania major promastigotes, but not with noninfective promastigotes obtained from log phase cultures. The antibody recognized a cell surface and released molecule that could be metabolically labeled with [14C]glucose, [3H]mannose, [3H]galactose, and [3H]palmitic acid, but not with [35S]methionine or [3H]leucine. The molecule was the major species surface-labeled by [3H]sodium borohydride after periodate treatment. The glycolipid appeared to be shed primarily as free carbohydrate because 70% of the released material partitioned in the aqueous fraction after phase separation in TX-114. The molecule could be distinguished from the L. major glycolipid which has already been extensively described because its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was of higher relative m.w. However, a close relationship between the two molecules was indicated by the finding that another monoclonal antibody, WIC-79.3, recognized both forms of the glycolipid; one produced and released only by log phase promastigotes, and one produced and released only by metacyclic promastigotes. The loss of agglutination with peanut agglutinin which has been shown to accompany metacyclogenesis was found to be caused by the loss of expression of the log form of the glycolipid which in most cases appeared to be the result of the developmental modification of this molecule. A survey of a number of virulent and avirulent. L. major strains and clones reinforced an absolute association between the ability of these promastigotes to initiate infection in BALB/c mice and their expression and release of the 3F12-binding, developmentally regulated form of the glycolipid. Not only does this glycolipid serve as the first well defined molecular marker for infective stage metacyclic promastigotes, but its unique structure is very likely to contribute to the adaptive changes that allow these parasites to survive within the vertebrate host.     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

Implications of disaggregation procedures on biological representation of human solid tumours

Abstract. This study was designed to define some biological aspects of cell suspensions, obtained by mechanical or enzymatic disaggregations, and to verify whether single cell suspensions are representative of original solid tumours. The study was performed on a series of 25 human solid tumours including breast carcinoma, ovarian carcinoma and malignant melanoma. A higher cell viability and a loss of aneuploid subpopulations, or a lower fraction of aneuploid cells, were observed in enzymatically-released samples than in samples obtained by the mechanical procedure. Moreover, the proliferative activity, which was generally similar for the cell suspensions obtained by the two disaggregation procedures, was always markedly lower in the cell suspensions than in solid samples from the same tumour. In conclusion, the results from this study indicate that many changes, such as selective release of cell populations from the tumour matrix, damage and destruction of aneuploid and proliferating cells can be induced to various extents by different disaggregation procedures.     

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-O-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion Channels in Southern Bean Mosaic Virus Capsid

The study of southern bean mosaic virus protein coat high resolution model revealed a structure with properties of a natural protein-ion channel. Coat protein pentamers form a 30-Å long channel and the amino acid composition of its wall bears some homology with the pentameric structure proposed for the nicotinic acetylcholine receptor channel. Ion transport properties were analyzed by computing ion-protein interaction energies on the basis of quantum chemistry methods. Energy maps show a channel attractive for cations, fully permeable to Li⁺ and a narrow barrier for other cations and water. The energy profiles found are similar to the profiles determined for the K⁺ channel of the sarcoplasmic reticulum. Comparisons with other icosahedral virus structures, including picornaviruses, suggest that ion channels would be a common feature of viral capsids. Biological roles for these channels are proposed.     

Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.