Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

Acceptor-donor relationships in the transglutaminase-mediated cross-linking of lens beta-crystallin subunits

Following the isolation of the N epsilon-(gamma-glutamyl)lysine-containing polymers from human cataracts, our efforts were directed to induce such cross-links experimentally in rabbit lens, and evidence was obtained for the selective reactivities of certain beta-crystallin subunits in this transglutaminase-catalyzed event. In the present work, we examined the enzymatic cross-linking of purified crystallins individually (alpha, beta H, beta L, and gamma) and in combinations, with particular emphasis on forming the approximately 55K dimer. This species was the primary product in the cross-linking of beta H-crystallins; beta L also reacted with transglutaminase. Neither alpha- nor gamma-crystallins formed appreciable amounts of cross-linked structures with transglutaminase. Dansylcadaverine, known to compete against the reactive lysines of proteins in forming N epsilon-(gamma-glutamyl)lysine cross-bridges, was shown to inhibit the generation of dimeric and higher ordered oligomers from beta H and beta L. The fluorescent amine specifically labeled only two subunits in beta H (approximately 29-30K and approximately 26K) and one in beta L (approximately 26K), identifying these substrates as possessing transglutaminase-reactive endo-gamma-glutaminyl residues. An antiserum to bovine beta Bp recognized the approximately 23K subunit of rabbit beta-crystallins and also the approximately 55K dimer, suggesting that the approximately 23K protein participates as a lysine donor in generating the cross-linked dimer with transglutaminase. Inasmuch as the same antiserum reacts with an approximately 50K material reported to appear in increasing amounts with age in human lens, the results lend added support to the physiological significance of transglutaminase in the aging of lens.     

Light and electron microscopical localization of concanavalin A lectin binding sites in rat epiphyseal chondrocytes

Summary Concanavalin A lectin binding sites have been detected within the cytoplasm of epiphyseal chondrocytes. Correlative light and electron microscopic results were obtained, indicating the presence of-d-mannose and/or -D-glucose residues detected by the lectin in the rough endoplasmic reticulum region. Quantitation of the electron microscopic cytochemical reaction also showed that the specific labelling was almost exclusively localized in the lumen of endoplasmic reticulum cisternae. No significant staining was found in other membrane compartments or extracellular matrix. This labelling pattern could be considered as the cytochemical evidence ofN-glycosylation processes occurring during the biosynthesis of cartilage extracellular matrix components by chondrocytes.     

Experimental study of the influence of errors of genetic correlation estimates on unrestricted,optimum, and desired gains selection indices

Summary Effects of errors in estimates of the genetic correlation on the accuracy of unrestricted, optimum, and desired gains selection indices were examined experimentally in Tribolium castaneum. Three lines were selected for three generations for pupal weight at 21 days and adult weight at 31 days, using unrestricted (I9), optimum (O9), and desired gains (G9) index selection methods. The genetic correlation between pupal and adult weights in the base population was 0.95. The optimum index was designed to set the response of pupal weight by a fixed amount, while in the desired gains index the responses of pupal and adult weights were specified as being equal to 31. Three other indices were constructed using a deliberately incorrect genetic correlation (0.25), i.e., unrestricted (I2), optimum (O2), and desired gains (G2). Responses observed in unrestricted index lines (I9 versus I2) and optimum index lines (O9 versus O2) did not differ significantly, even though lines I9 and I2 differed in a practical sense. Responses in desired gains index lines (G9 versus G2) differed significantly. Responses obtained for aggregate genotype (pupal weight + adult weight) and for the component traits were greater in line I9 than those obtained in line I2. Responses obtained in the O9 and O2 lines for pupal and adult weights were similar, while those obtained in the G9 and G2 lines were similar for pupal weight but not (P<0.05) for adult weight. Therefore, underestimation of the genetic correlation seems to affect the efficiency of a desired gains index more than that of unrestricted or optimum indices.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

Serotonin uptake in the central nervous system of rats fed a corn-diet

1. Endogenous serotonin (5-HT), 5-hydroxyindol acetic acid (5-HIAA) content and exogenous 5-HT uptake (Km and Vmax) were measured in different brain regions (cerebellum, diencephalon, brain stem and telencephalon) of rats fed with a corn diet and restricted protein (8%) diet during 6 weeks. 2. A reduction of 5-HT levels was found in all regions studied of animals fed a corn diet, whereas, 5-HIAA was only decreased in brain stem and diencephalon. 3. An important increase in Km and Vmax were registered in brain stem and diencephalon of protein restricted animals, whereas, an increase of 5-HT uptake affinity in cerebellum, brain stem and telencephalon (35, 42 and 33% respectively) was observed. Simultaneously, under corn diet conditions, the Vmax decreased 40, 30 and 34% respectively in those regions. 4. It is suggested that the brain stem was the more sensitive area under nutritional restricted conditions and the development of some possible compensatory mechanisms of the 5-HTergic system is discussed.     

Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

Formation of a 55 000-weight cross-linked beta crystallin dimer in the Ca2+-treated lens. A model for cataract

Incubation of lens in Ca2+-containing media, considered by several investigators to be a useful model of cataract formation, gave rise to significant alterations in the covalent structures of various proteins. In rabbit lens, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used after reduction of disulfides in urea, the most readily observable changes were (i) disappearance of 210K, 95K, and 60K proteins, (ii) modifications of alpha crystallin subunits, (iii) alterations of beta H crystallins, and (iv) de novo production of 55K and higher molecular weight polymers. The addition of leupeptin inhibited the disappearances of 210K, 95K, and 60K proteins and the alteration of alpha crystallins, suggesting that all these were caused by a Ca2+-activated protease. The proteolytically sensitive 60K species was identified as vimentin, a component of intermediate filaments. Formation of the 55K material and of higher molecular weight polymers during Ca2+ treatment of the lens could be prevented by histamine, a compound known to inhibit the transglutaminase-mediated cross-linking of proteins by epsilon-(gamma-glutamyl)lysine peptide bonds in other biological systems. It could also be shown by immunoblotting that an antibody raised against the 55K material reacted selectively with beta crystallins of normal lens. This indicates that the 55K product is in all likelihood an essential intermediate toward higher polymers and that the 55K product is a cross-linked dimer of certain polypeptides of beta crystallin.(ABSTRACT TRUNCATED AT 250 WORDS)