Dimethyl suberimidate as a specific inductor of apoptosis in transformed cells

A modification of protein-protein interactions can be considered to be a way to regulate cell death. Chemical cross-linking agents have been traditionally used for protein complexing. This study has been undertaken to test a possibility to induce and(or) to modify cell death by a homobifunctional cross-linker dimethyl suberimidate (DMS). It was shown that the protein cross-linking by DMS resulted in a death of transformed cells by apoptosis. DMS-induced apoptosis was accompanied by cell cycle perturbations and down-regulation of p21/Waf1 mRNA expression. The RT-PCR analysis of bcl-2 family genes revealed the engagement of mitochondria in DMS-induced cytotoxicity. Then, the influence of DMS treatment on TNF-dependent and Fas-mediated apoptosis was investigated. Cell pre-incubation with DMS resulted in their increasing sensitivity for the TNF cytotoxic effect, though activities of anti-Fas cytotoxic antibodies were inhibited. The effects observed are probably due to cross-linking of TNF-receptors. Thus, this study first demonstrated that a chemical cross-linker DMS in capable of inducing apoptosis in transformed cells and modifying TNF-dependent and Fas-mediated apoptosis.     

The human platelet as an independent unit for sulfate conjugation

The human platelets possess a full complement of enzymes capable of synthesizing N-acetyldopamine (NADA) 35sulfate from ATP, Mg++ and sodium 35sulfate. The pH optimum for this three-step overall sulfate conjugation (comprising of the ATP sulfurylase, APS kinase and phenolsulfotransferase reactions) is 8.6 and the reactions proceeded progressively for several hours. Both ATP and Mg++ ions, above their respective optimal concentrations of 5 and 7 mM, inhibited the sulfate conjugation of NADA. The apparent Km values for NADA as determined by the phenolsulfotransferase (PST) and overall reactions were similar in magnitude being 2.6 and 4.8 microM, respectively, while that for sodium 35sulfate was 202 microM. A comparison of these two activities in 62 platelet preparations of normal subjects showed that the rate of the PST reaction was generally higher than the overall reaction even though the PST assay was carried out at suboptimal concentration of PAPS. There was a positive correlation (r = 0.82) between the two sets of data, suggesting that the PST reaction probably has some control over the rate of overall sulfate conjugation.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

V-ATPase as an effective therapeutic target for sarcomas

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern.     

The antibody-enzyme analogy. Comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine.

Reduced Schiff base compounds of pyridoxal-P and tyrosine, which were used to induce specific antibodies described in the preceding article (V. Raso and B. D. Stolar, Biochemistry, 1975), caused active site-directed inhibition of tyrosine transaminase and tyrosine decarboxylase. The antibodies, studied as analogs of enzymes, were able to bind an unsaturated Schiff base catalytic intermediate, as shown by equilibrium dialysis and absorbance difference spectroscopy. Schiff base formation can proceed while the pyridoxal-P and tyrosine are within the antibody combining site, but the rate of this bimolecular condensation within the sites was not greater than the rate in free solution. Antibody did effect a small rate enhancement for the pyridoxal-P-catalyzed transamination of L-tyrosine. These results are discussed in light of current ideas in the mechanisms of enzyme catalysis.     

Application of 2-amino-6-vinylpurine as an efficient agent for conjugation of oligonucleotides

Attempts have been made to conjugate a variety of molecules with oligonucleotides to achieve useful functions. In this study, we have established a new efficient method for post-synthetic conjugation of oligonucleotides with the use of the 2-amino-6-vinylpurine nucleoside. Amino nucleophiles form the corresponding conjugates under acidic conditions, whereas thiol nucleophiles reacted efficiently under alkaline conditions. Thus, glutathione and HS-Cys-(Arg)8 without protecting groups were efficiently conjugated to the 2-amino-6-vinylpurine-bearing ODN under alkaline conditions. The use of 2-amino-6-vinylpurine as an agent for conjugation is advantageous in that it is stable during the reaction and may be applied to conjugation of ODNs with multiple functional molecules.     

A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer

Cowpea mosaic virus (CPMV), an icosahedral 30 nm virus, offers a uniquely programmable biological nanoscaffold. This study reports initial optimization of the simultaneous modification of two CPMV mutants with AlexaFluor 647 fluorescent dyes and either IgG proteins or antibodies at specific sites on the virus scaffold. The capacity of CPMV as a simultaneous carrier for different types of molecules was demonstrated, specifically, when applied as a tracer in direct and sandwich immunoassays. The ability to label the virus capsid with antibody and up to 60 fluorescent dyes resulted in an improved limit of detection in SEB sandwich immunoassays, when used as a tracer, relative to a mole equivalent of dye-labeled antibody.     

Soybean peroxidase as an effective bromination catalyst*

Soybean peroxidase (SBP), an acidic peroxidase isolated from the seed coat, has been shown to be an effective catalyst for the oxidation of a variety of organic compounds. In the present study, we demonstrate that SBP can catalyze halogenation reactions. In the presence of H(2)O(2), SBP catalyzed the oxidation of bromide and iodide but not chloride. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) served as a useful substrate for SBP-catalyzed halogenations yielding the 6-bromo derivative. Halogenation of veratryl alcohol was optimal at pHs below 2.5 with rates of 2.4 μm/min, achieving complete conversions of 150-μm veratryl alcohol in 24 h. The enzyme showed essentially no brominating activity at pHs above 5.5. SBP-catalyzed bromination of veratryl alcohol proceeded with a maximum reaction velocity, (V(max))(apparent), of 5.8 x 10(-1) μm/min, a K(m) of 78 μm and a catalytic efficiency (k(cat)/K(m) of 1.37 x 10(5) M/min at pH 4.0, assuming all of the enzyme's active sites participate in the reaction. SBP also catalyzed the bromination of several other organic substrates including pyrazole to produce a single product 1-bromopyrazole, indole to yield both 5-bromoindole and 5-hydroxyindole, and the decarboxylative bromination of 3,4 dimethoxy-trans-cinnamic acid to trans-2-bromo-1-(3,4 dimethoxyphenyl)ethylene. A catalytic mechanism for SBP-catalyzed bromination has been proposed based on experimental results in this and related studies.     

Open communication as an effective stress management method for breast cancer patients

The social and emotional adaptation of 51 breast cancer patients was assessed four times during the first year after mastectomy according to Weissman and Paykel's Social Adaptation Scale (SAS) and judges' ratings. Openness of communication was measured by eight indices during the first two interviews. It was predicted that they would correlate positively with successful adaptation as measured at the third and fourth interviews. Most patients were aware of their diagnosis, and their communication about their plight was found to be multifaceted. Successful copers sought information; less successful copers avoided it. However, where emotions and not facts were the issue, palliative measures such as avoidance of speaking about the threat and refusal to accept its further implications were connected with better adjustment. The findings indicate that palliation is a prerequisite to good instrumental adjustment when the emotional reactions are intense and countermeasures are limited. More research is needed for assessing communication with specific others and change over time.     

Potentiality of tricyclic compound thioridazine as an effective antibacterial and antiplasmid agent.

Thioridazine (Th), which is therapeutically used in psychiatric patients, was found to possess conspicuous antimicrobial activity when tested against 316 strains belonging to a number of Gram positive and Gram negative bacteria. Although Staphylococcus aureus, Vibrio chloerae and V. parahaemolyticus were found to be most sensitive, Th was highly bactericidal against S. aureus and bacteriostatic for vibrios and other Gram negative organisms. In the study of antiplasmid/curing effect of Th on twelve multiply antibiotic and Th resistant bacteria, it was observed that elimination of R plasmids was facilitated by choice of optimal concentration of Th. Significant elimination of single and combined antibiotic resistance occurred in E. coli and Shigella flexneri and not in S. aureus.     

Tumor vascular targeting using a tumor-tissue endothelium-specific monoclonal antibody as an effective strategy for cancer chemotherapy.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.     

Seed P-enrichment as an effective P supply to wheat

Most fertilizer phosphorus (P) is sorbed by soil rather than being taken up by crops. We hypothesize enriching wheat seed with P before sowing the crop will reduce requirement of fertilizer P for subsequent wheat production. We produced P-enriched wheat (Triticum aestivum L.) seed by soaking the seed in different concentrations of potassium phosphate solution. We found that ~0.35 M potassium phosphate was the most effective concentration for P-enrichment of the seed. In pot and field experiments we found that the P-enriched seed required ~60% less fertilizer P than seed not soaked with potassium phosphate before sowing. Increases in shoot P content could not be explained only by the increase of seed P-enrichment, suggesting that P acquisition from soil was also enhanced. Under hydroponic conditions we found that root length was greater in seedlings grown from P-enriched seed with higher specific root length than in seedlings grown from non-P-enriched seed. We conclude P-enrichment of wheat seed before sowing reduces fertilizer P requirements of plants.     

Low-dose zinc administration as an effective Wilson's disease treatment

A case of a 11-yr-long Wilson’s disease treatment in a 16-yr-old boy with neurologic presentation was analyzed and monitored. In the face of severe symptoms of chelator intolerance, a comparatively low dose of 100 mg of zinc has been administered for the entire 11-yr-long treatment. Considerable improvement of clinical status was achieved, with accompanying regression of central nervous system lesion. The parameters of copper metabolism were normalized with effective urine elimination. The low-dose oral zinc intake proved to be therapeutically effective, eliminating further copper tissue toxicity.