Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Sigma receptors in schizophrenic cerebral cortices

Antipsychotics represent high affinity for sigma receptors and sigma-like drugs often have the psychotomimetic properties. Besides, the receptors are unevenly distributed in human brain. These findings suggest that sigma receptors might be involved in the pathophysiology of schizophrenia. Sigma receptors in rat and human brain were measured with [³H]-1, 3, di-o-tolylguanidine (DTG) and non-specific binding of [³H]DTG was determined in the presence of 10^–5M haloperidol. Monovalent and divalent cations strongly inhibited [³H]DTG binding. Glutamate, aspartate and glycine also decreased the binding to human cerebral membranes. With post-mortem brain samples from 12 schizophrenics and 10 controls, sigma receptors were measured in 17 areas of cerebral cortex. Sigma receptors binding showed the regional differences in the cortex, but no significant differences between schizophrenics and controls were observed except the superior parietal cortex where the binding significantly increased in the schizophrenic group. These results suggest that sigma receptors in cerebral cortices might not be directly concerned with the pathophysiological role in schizophrenia.Dedicated to Dr. Morris Aprison. Received too late for publication in special issue.     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Outer membranes of Gram-negative bacteria are permeable to steroid probes

The permeability of bacterial outer membranes was assayed by coupling the influx of highly hydrophobic probes, 3-oxosteroids, with their subsequent oxidation catalysed by 3-oxosteroid delta 1-dehydrogenase, expressed from a gene cloned from Pseudomonas testosteroni. In Salmonella typhimurium producing wild-type lipopolysaccharide, the permeability coefficients for uncharged steroids were 0.45 to 1 x 10(-5) cm s-1, and the diffusion appeared to occur mainly through the lipid bilayer domains of the outer membrane. These rates are one or two magnitudes lower than that expected for their diffusion through the usual biological membranes. The permeation rates were markedly increased (up to 100 times) when the lipopolysaccharide leaflet was perturbed either by adding deacylpolymyxin or by introducing mutations leading to the production of deep rough lipopolysaccharides. An amphiphilic, negatively charged probe, testosterone hemisuccinate, penetrated much more slowly than the uncharged steroids. Study of various Gram-negative species revealed that P. testosteroni, Pseudomonas acidovorans, and Acinetobacter calcoaceticus showed higher outer membrane permeability to steroid probes and higher susceptibility to hydrophobic agents such as fusidic acid, novobiocin and crystal violet relative to S. typhimurium and Escherichia coli.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.