Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Biochemical similarity among serologically distinct flagellins of Campylobacter jejuni

Abstract We describe a simplified method for obtaining highly purified flagellin, suitable for biochemical analysis using HPLC-gel permeation. Amino acid composition and N-terminal sequence analyses were performed on flagellins from serologically distinct isolates. The amino acid composition of flagellin from 10 strains was very similar. The N-terminal amino acid sequence is highly conserved. Significant sequence homology was found with flagellin of Bacillus subtilis .     

Growth,aluminium uptake and mucous cell morphometrics of early life stages of brown trout,Salmo trutta,in low pH water

Synopsis Young (7–10 days after hatching) brown trout (Salmo trutta) exposed for 5 days to pH 5 in high calcium water and at 2 temperatures (12°, 4°C) in the laboratory displayed no alterations in growth or in mucous cell concentration and volume, compared to the control group kept at pH 7.2. Contamination of acid-stressed young with 230 μg All^-1 resulted in significant growth depression and Al accumulation, but in no changes of mucous cell morphometrics. Field tests in low calcium water produced high mortality at low pH (5.1), but showed consistent effects on mucous cells as in laboratory experiments. Three-month-old juveniles of brown trout, subjected to decreased pH values at 12° and in high calcium water for 8 days exhibited mucous cell hyperplasia (without hypertrophy) within 3 h of the acid addition. After 120 h sloughing of the integument occurred with full recovery not possible within a 4-day-recovery period. Although the results presently apply only to hard water conditions, the differences between juveniles and recently hatched young in tolerance to pH- and Al-mediated stress may also be of importance for soft waters affected by acid rain.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

Growth and production of Euphausia lucens in the southern Benguela Current

The growth rate of a population of Euphausia lucens from thewest coast of South Africa was estimated from laboratory studiesand from monthly size-frequency distributions of samples collectedover a 1-year period. Laboratory studies indicated that growthrates ranged from 0.131 (larvae) to 0.047 mm day^–1 (juveniles),while size-frequency distributions suggested a growth rate of{small tilde}0.026 mm day^–1 for the adults. The mean annualbiomass from the inshore, intermediate and offshore regionsranged from 9.75 to 47.29 mg dry wt m^–3 with the highestbiomass being found in the inshore region. Calyptopis larvaewere present for most months of the year, indicating continuousrecrwtment. The relative contribution of flesh, moults and eggsto the total annual production was estimated separately forall three regions. Production due to growth (P_g) was estimatedto be 92.71–185.60 mg dry wt m^–3 year^–1, whileexuviai production (P_e) varied between 60.01 and 281.38 mg drywt m year Production of eggs (P_r) was estimated to range from5.07 to 12.39 mg dry wt m year the lowest value being obtainedin the inshore region. Moult production represented {small tilde}6times the mean biomass in each region, while the P/B ratio forflesh production varied from 3.92 to 8.91, the highest ratiobeing obtained in the offshore region. Total P/B ratios rangedfrom 10.14 to 16.01.