Inhibition of arachidonate biosynthesis in hepatoma tissue culture cells by 11-deoxycorticosterone-induced factor

In this work it was demonstrated that the incubation of hepatoma cultured cells (HTC 7288 c) with 11-deoxycorticosterone (DOC) ranging from 0 to 10^–4M concentration provoked a dose-dependent inhibition in the conversion of [1^–14C] eicosatrienoic acid to arachidonic acid. This steroid also produced an increase in the uptake of exogenous 20: 3 (n-6) acid. The depressive effect evoked by DOC on 5 desaturating activity was reflected on the fatty acid composition changes of the hepatoma cells. The 5 desaturase activity was inhibited by a soluble factor that would be induced by the hormone and that was present in the cytosol fraction from DOC-treated cells, corresponding to a low molecular mass below 25 kDa. Presently we report that an 11--OH group on the steroid molecule is not an essential requirement for the production of a 5 desaturase inhibitory factor.Members of the Carrera del Investigador Científico, CONICET, Argentina     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Amelanchier alnifolia vegetation in eastern Idaho,USA and its environmental relationships

Fifteen shrub stands of Amelanchier alnifolia vegetation were studied using Braun-Blanquet procedures, tabulated, and ordered by Twinspan, Decorana, and field experience into 5 groups of stands. The stands occurred on the lower slopes of the southwestern Teton and southeastern Big Hole mountains of eastern Idaho and in-and outside a U.S. Forest Service exclosure above the Hoback River in adjacent Wyoming. The ecology of this Amelanchier vegetation as a whole and of the 5 groups of stands is discussed using Jenny's factors of soil formation (1941, 1958, 1980) as a framework. Plant indicator values (Ellenberg 1979, 1988) which rank species on their occurrences in relation to light, temperature, continentality of climate, water availability, soil reaction (pH), and soil nitrogen supply, were independently assigned to the 121 species of vascular plants encountered in the described stands.     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Role of fatty acids of (n-3) and (n-6) series on α-linolenic acid desaturation and chain elongation in HTC cells

Summary The desaturation and chain elongation of [1-¹⁴C] -linolenic acid were studied in HTC cells preincubated for 24 h in the presence of different unlabeled fatty acids of (n-3) and (n-6) series. After 24 h in the presence of [1-¹⁴C] -18:3, cells transformed this acid into labeled 20:5 and 22:5(n-3) through the desaturation-elongation pathway and into 20:3 and 22:3(n-3) by the elongation reactions. The preincubation of HTC cells with (n-3) fatty acids (-18:3, 20:5 and 22:6) produced an increase in the amount of [1-¹⁴C] -18:3 that remained in the cells without being metabolized and consequently, a decrease in the last product formed, the 22:5(n-3) was observed. Simultaneously, the desaturation-elongation products decreased significantly and those of the elongation pathway were not modified, except when the cells were pre-incubated with the last fatty acid of this family (22:6) which increased this metabolic route. Fatty acids of (n-6) series (-18:3, 20:3, 20:4 and 22:4) decreased the desaturation-elongation pathway and increased the elongation route from [1-¹⁴C] -18:3. From these results, it can be concluded that fatty acids of (n-3) family and intermediates of (n-6) series would impair the [1-¹⁴C] -18:3 metabolism at the 6 desaturation step. The fatty acid composition of the cells was also modified by the preincubation with (n-3) and (n-6) acids showing a decrease on 9 desaturation activity.     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

Fatty acid utilization by young Wistar rats fed a cafeteria diet

The content and accretion of fatty acids in 30, 45 and 60-day old Wistar rats fed either reference chow or a cafeteria diet has been studied, together with their actual fatty acid intake during that period. Diet had a small overall effect on the pattern of deposition of fatty acids, but the deposition of fat was much higher in cafeteria rats. The fat-rich cafeteria diet allowed the direct incorporation of most fatty acids into lipid storage, whilst chow-feeding activated lipogenesis and the deposition of a shorter chain and more saturated type of fatty acids. During the second month of the rat's life, the elongation pathway as well as ⁹-desaturase became functional, thus helping to shape the pattern of fatty acids actually accrued. The 60-day rats showed a relative impairment in the operation of ⁵-desaturase, since their lipids had a higher C20:4/C20:3 ratio than those of the diet ingested. Cafeteria-diet feeding minimized this effect since the large supply of dietary polyunsaturated fatty acids made the operation of the elongation-desaturase pathways practically unnecessary.     

Continuous production of 5′-ribonucleotides from yeast RNA by hydrolysis with immobilized 5′-phosphodiesterase and 5′-adenylate deaminase

The production of 5-IMP and 5-GMP by enzymatic conversion from RNA using a continuous two packed-bed reactor was investigated. 5-Phosphodiesterase (5PD) and 5-adenylate deaminase (5AD) were immobilized in an acrylic resin to produce derivatives with about 15 U/g of support. The kinetic properties of the enzymes were described by Michaelis-Menten models: no significant differences were found in the K _m value of the free and immobilized 5AD (60 and 20 m, respectively), whereas for 5PD the K _m value was one order of magnitude higher for the immobilized enzyme (4.85 mg RNA/ml), probably due to diffusional limitations. Both enzymes remained stable after 8 h of use in a continuous packed-bed reactor whereas the half lives of the free enzymes were 193 min and 240 min at 40°C and 70°C for 5AD and 5PD, respectively. A procedure is proposed for the design of a continuous two packed-bed column process.F. Olmedo and F. Iturbe are with the Depto. de Alimentos y Biotecnologia, Facultad de Química, UNAM, México 04510, D.F., Mexico. J. Gomez-Hernández is with the Depto. de Biotecnología, UAM-1, Apdo. Postal 55-535, México 09340, D.F., Mexico. A. López-Munguía is with the Instituto de Biotecnología, Apartado Postal 510-3, Cuernavaca, Mor. 62271, Mexico     

Antimutagenic Role of Autonomous 3′ → 5′-Exonucleases

Original and published data on the antimutagenic role of autonomous 3 5-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but are often involved in replicative complexes. AE overproduction in bacterial cells is accompanied by a sharp suppression of mutagenesis, whereas AE inactivation in bacteria and higher fungi results in the increase in mutation rates by two to three orders of magnitude. The combined action of AE and DNA polymerases substantially improves the fidelity of their functioning in vitro. The fidelity of nuclease-free DNA polymerases and increases by two to three orders of magnitude in the presence of AE. The fidelity of moderately processive DNA polymerase I increases by two orders of magnitude, and that of highly processive DNA polymerase increases by a factor of 5–10, although both these polymerases possess their own 3 5-exonucleolytic activity. In biochemical experiments, AE was shown to participate directly in the correction of errors made by DNA polymerase I. The presence of AE in multienzyme DNA polymerase complexes increases their fidelity by a factor of 5–10. A model of extrinsic proofreading by AE in DNA biosynthesis is proposed. An investigation of thirty objects from all three kingdoms of life (from archaea and bacteria to mammals, including humans) has shown that AE account for 30–90% of the total cellular 3 5-exonucleolytic activity. Therefore, AE increase significantly the intracellular ratio of 3 5-exonuclease to DNA polymerase activities in a wide phylogenetic variety of species, which always leads to the increasing fidelity of DNA biosynthesis.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Fatty acid Δ5 desaturation in rat liver cell nuclei

Activity of one of the key enzymes involved in arachidonic acid (20:4 n–6) biosynthesis, the 5 desaturase, was found in rat liver cell nuclei. Up to now, it has been shown that the fatty acid desaturases are located exclusively in the endoplasmic reticulum. Similarly to what happens with microsomal enzyme the nuclear 5 desaturase enzyme was only fully active in the presence of a cytosolic factor. In this condition it reached a specific activity of 50 pmol 20:4 n–6 formed/min/mg of protein. This fact would imply that purified nuclei like purified microsomes lack a soluble cytosol factor necessary for the total desaturation reaction expression. Besides the nuclear 5 desaturase has an optimal pH of 7.6 and is inhibited by 1 or 10 mM KCN. Low long chain acyl-CoA synthetase activity that catalyzes the formation of 20:3 n–6-CoA, was also found in liver nuclei. This step would be essential in nuclear desaturation since when ATP and/or CoA (necessary for the acylation reaction) are omitted from the incubation mixture, the desaturation reaction does not take place.Abbreviation PMSF phenylmethylsulfonyl fluoride     

The origin of crop weed communities composed of summer annuals

Summary Evidence for the origin and dating of crop weed communities composed of summer annuals is to be found in palaeobotanical and archaeological data. There is archaeological evidence that around the middle of the first millennium BC fields were taken into permanent cultivation, even on the poorer soils.Fertilization with lime and manure and Plaggenwirtschaft were evidently involved in this early agriculture. In this paper the idea is put forward that Plaggenwirtschaft resulted in a sudden increase in the number of weed species brought together in cornfields and that this practise led to the very first beginning of our modern weed communities.Translated into English by Drs. C. van Driel-Murray.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

The freshwater microcrustacea of Easter Island

The Cladocera, Copepod and Ostracod fauna of Easter Island amounts to only five species. Three of these are wide-ranging, and four are cyclic parthenogens or at least capable of parthenogenesis. Two, the Cladoceran Alona weinecki and the Ostracod Sarscypridopsis sp., are more interesting from a biogeographic point of view, because restricted (apart from Easter Island) to the subantarctic. It is argued that this is strong evidence of their introduction by man, not by natural passive dispersal.