The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5''-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Bombesin stimulates inositol polyphosphate production in GH4C1 pituitary tumor cells: comparison with TRH

The hormones bombesin and thyrotropin-releasing hormone (TRH) stimulated formation of inositol- monophosphate, bisphosphate, trisphosphate and tetrakisphosphate with parallel time courses in GH4C1 cells, while a more polar inositol polyphosphate peak, consisting of inositol-pentakisphosphate and perhaps also inositol-hexakisphosphate, was unaffected by either hormone. Although bombesin and TRH had similar potencies in stimulating inositol trisphosphate production (Km = 30 nM and 40 nM, respectively), TRH was significantly more efficacious than bombesin. Maximal stimulation of inositol-1,4,5-trisphosphate formation by TRH was not further increased by addition of a maximally effective dose of bombesin, suggesting that the two hormones act through stimulation of a common pool of phospholipase C, and this enzyme pool can be fully stimulated by TRH, alone.     

Possible role of the membrane Na+/H+ antiport in ornithine decarboxylase induction by L-asparagine

Amino acids of transport systems A and N play certain important role in cell activation. For example, the presence of these amino acids is essential in the induction of ornithine decarboxylase by growth factors and hormones. At mM concentrations, each of these amino acids, particularly L-asparagine, can also induce the enzyme without being further metabolized or incorporated into proteins. We have reported that the addition of 10 mM L-asparagine to quiescent Reuber's H-35 rat hepatoma cells caused an immediate and transient increase in intracellular pH. Here we report that concomitant with the intracellular alkalinization was an increase in H+ extrusion which was amiloride-sensitive and Na+-dependent. The induction of ornithine decarboxylase by L-asparagine was also amiloride-sensitive.     

Effects of butylated hydroxytoluene on membrane lipid fluidity and freeze-thaw survival in mammalian cells

Butylated hydroxytoluene (BHT) increases the fluidity of membrane lipids in the hydrocarbon but not the polar regions, as measured by electron spin resonance spin label probes. BHT also sensitizes nucleated mammalian cells to freeze-thaw damage as measured by colony formation survival assays. Furthermore, the membranes of BHT-exposed cells are more susceptible to physical stress, as reflected by the BHT-induced sensitization to hypotonic stress. Since others have shown that BHT induces hexagonal phase lipids in lipid bilayers, this phenomenon may also influence the above survival results.     

Modulation of Affinity of a Marine Pseudomonad for Toluene and Benzene by Hydrocarbon Exposure

Trace (microgram liter⁻¹) quantities of either toluene or benzene injected into an amino-acid-limited continuous culture of Pseudomonas sp. strain T2 were utilized immediately with affinities of 2.6 and 6.8 liters g of cells⁻¹ h⁻¹, respectively, and yielded large amounts of organic products, carbon dioxide, and cells. The immediate utilization of hydrocarbons by hydrocarbon-deprived organisms helps to establish the nutritional value of nonpolar substrates in the environment. The observation of small Michaelis constants for toluene transport led to tests of metabolic competition between hydrocarbons; however, competitive inhibition of toluene metabolism was not found for benzene, naphthalene, xylene, dodecane, or amino acids. Benzene and terpenes were inhibitory at milligram liter⁻¹ concentrations. Toluene was metabolized by a strongly inducible system when compared with benzene. The capacity of toluene to effect larger affinity values increased with exposure time and concentration. The kinetics of induction suggested saturation phenomena, resulting in an induction constant, K_ind, of 96 μg of toluene liter⁻¹. Maximal induction of amino-acid-grown cells required about 80 h, with the affinity reaching 317 liters g of cells⁻¹ h⁻¹.     

On the evolution of non-specific mutualism

It has been argued that mutualisms are non-specific when mutualistic interactions are weak and transient, and become more specific as interactions increase in strength. However, this runs counter to the observation that there exist tightly linked mutualisms of great antiquity that are highly nonspecific. Here we argue that mutualism generates positive, interspecific, frequency-dependent selection, which acts as a cohesive evolutionary force, discouraging evolution of specificity. A simple mathematical model is constructed to analyse the evolution of a community consisting of two guilds of species with mutualistic between-guild interactions, two competing species in each guild and two genetically distinct phenotypes within each species. With some simplifying assumptions, the trajectories in the neighbourhood of the only interior equilibrium point are determined analytically in terms of interactions between individuals. These show that the equilibrium is locally stable (no evolution) when there is little differentiation between phenotypes in mutualistic and interspecific, competitive interactions. On the other hand, when there is strong differentiation between phenotypes in their mutualistic interactions, the equilibrium is unstable and the community starts to evolve towards non-specificity. There are, however, two forces counteracting this tendency which, if sufficiently potent, cause evolution towards specificity. The first is generated by strong differentiation between phenotypes in interspecific competition; the second is caused by specificity which already exists between species in their mutualistic interactions. Thus, the tendency for non-specificity or specificity to evolve depends on the interplay between antagonistic and mutualistic interactions in the community. We illustrate these results with some numerical examples and, finally, survey some data on specificity of mutualisms in the light of the analysis.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.