Effect of n-3 and n-6 fatty acids on hepatic microsomal lipid metabolism: a time course study

The present study examines the time dependent effects of n-6 and n-3 polyunsaturated fatty acids on liver microsomal lipid metabolism in FVB mice fed a diet supplemented with a mixture of free fatty acids (mainly 18:3n-6 and 20:5n-3) at 25 mg/g diet. Significant changes in the fatty acid composition of total liver and microsomal lipids were observed after 7 days on the diets. Thereafter, some animals remained on the same diet while others were fed a diet supplemented with hydrogenated coconut oil (HCO). With the exception of 20:5n-3 which showed a slower recovery, establishment of the HCO pattern was rapid indicating that the diet-induced changes could be easily reversed. The unsaturation index, the cholesterol/phospholipid ratio and the microviscosity of the microsomal membranes were not affected by these dietary manipulations. Unsaturated fatty acid supplementation reduced the activity of ⁹ desaturase by 50%. Feeding the HCO diet to mice previously fed the EPA/GLA diet led to a progressive increase in ⁹ desaturase activity, reaching 80% of the day zero values after 14 days. The monoene content of hepatic total lipids reflected, in most cases, the changes in enzyme activity. This study shows that a low dose of a n-3 and n-6 free fatty acid mixture increases the quantities of members of the n-3 family, without loss of n-6 fatty acids in microsomal membranes and modifies the activity of ⁹ desaturase without altering the microsome physicochemical parameters.     

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.     

Phenolic Compound Utilization by the Soft Rot Fungus Lecythophora hoffmannii

Nine phenolic compounds were metabolized by the soft rot fungus Lecythophora hoffmannii via protocatechuic acid and subsequently cleaved by protocatechuate 3,4-dioxygenase as determined by oxygen uptake, substrate depletion, and ring cleavage analysis. Catechol was metabolized by catechol 1,2-dioxygenase. Fungal utilization of these aromatic compounds may be important in the metabolism of wood decay products.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.     

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.     

Orientation in a desert lizard (Uma notata): time-compensated compass movement and polarotaxis

Summary The diurnal escape response of fringetoed lizards (Uma notata) startled by predators demonstrates clear directional orientation not likely to depend on local landmarks in the shifting sands of their desert environment. Evidence that celestial orientation is involved in this behavior has been sought in the present experiments by testing the effects of (1) phase shifting the animal's internal clock by 6 h and (2) by training the lizards to seek shelter while exposed to natural polarization patterns. In the first case, 90° shifts in escape direction were demonstrated in outdoor tests, as expected if a time-compensated sun or sky polarized light compass is involved. In the second instance, significant bimodale-vector dependent orientation was found under an overhead polarizing light filter but this was only evident when the response data were transposed to match the zenithe-vector rotation dependent on the sun's apparent movement through the sky. This extends to reptiles the capacity to utilize overheade-vector directions as a time-compensated sky compass. The sensory site of this discrimination and the relative roles of sun and sky polarization in nature remain to be discovered.     

Mapping of brain activity in mesencephalic and diencephalic structures of toads during presentation of visual key stimuli: a computer assisted analysis of (14C)2DG autoradiographs

Summary The (¹⁴C)2DG autoradiographic technique has been employed to quantitatively map glucose utilization in the mesencephalon, the diencephalon and the cerebellum, of toads in response to configurational moving visual stimuli: (i) a 0.4 cm × 2.8 cm worm-like stripe (W) which elicited prey catching responses, (ii) a 8.4 cm × 8.4 cm square (S) that released predator avoidance responses, and (iii) a 2.8 cm × 0.4 cm antiworm-like stripe (A) which elicited no motor activity.For various brain nuclei different relationships were obtained: The optic tectum showed statistical significant higher 2DG uptake during worm-stimulation (¯X _W) than during antiworm stimulation (¯X _A), i.e.¯X _W>¯X _A. The latter visual pattern led to a 2DG utilization that was statistically significant stronger than during stimulation with a square (¯X _S), i.e.¯X _A>¯X _S. Thus, in comparison between right and left hemisphere as well as between brains the following ratios were obtained:Optic tectum:¯X _W>¯X _A>¯X _S; nucleus isthmi:¯X _W>¯X _A-¯X _s; posterodorsal lateral thalamic nucleus:¯X _S>¯X _A>¯X _W; posteroventral lateral thalamic nucleus:¯X _S>¯X _A¯X _W; posterior thalamic nucleus:¯X _W>¯X _A¯X _S; anteripr division of the lateral thalamic nucleus:¯X _W>¯X _A¯X _S; anterior thalamic nucleus:¯X _A>¯X _S>¯X _W; nucleus of Bellonci and dorsal division of the ventrolateral thalamic nucleus:¯X _W¯X _A¯X _S; cerebellum:¯X _S¯X _W>¯X _A.Abbreviations A anterior thalamic nucleus - Cb cerebellum - Hyp hypothalamus - Ist nucleus isthmi - cl. Ist contralateral Ist - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, posterodorsal division - Lpv lateral thalamic nucleus, posteroventral division - MP medial pallium - NB/VLd nucleus of Bellonci and ventrolateral thalamic nucleus, dorsal division - P posterior thalamic nucleus - PO preoptic area - Sna snapping evoking area=ventrolateral tectum - Str striatum - Tec tectum opticum     

Adjacent chromosomal regions can evolve at very different rates: Evolution of theDrosophila 68C glue gene cluster

Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.