Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serum-less conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.     

Inch by inch, row by row

Bacterial chemotaxis systems have cooperatively interacting clusters of transmembrane receptors and signaling proteins to detect, amplify, integrate and adapt to environmental signals. A recent study provides experimental data to construct a new model of the signaling complex.     

Chicks, like children, spontaneously reorient by three-dimensional environmental geometry, not by image matching

Spatial reorientation by layout geometry occurs in numerous species, but its underlying mechanisms are debated. While some argue that navigating animals' sense of place is based on geometric computations over three-dimensional representations, others claim it depends on panoramic image-matching processes. Because children reorient by subtle three-dimensional perturbations of the terrain and not by salient two-dimensional brightness contours on surfaces or freestanding columns, children's sense of place cannot be explained by image matching. To test image-matching theories in a different species, the present experiment investigates the reorientation performance of domestic chicks (Gallus gallus) in environments similar to those used with children. Chicks, like children, spontaneously reoriented by geometric relationships of subtle three-dimensional terrains, and not by salient two-dimensional brightness contours on surfaces or columns. These findings add to the evidence for homologous navigation systems in humans and other vertebrates, and they cast doubt on image-matching theories of reorientation in these species.     

Recent visitations by eels to Sable Island, Canada, confirmed by parasites

The presence of immature stages of Proteocephalus macrocephalus in nine-spined stickleback Pungitius pungitius , four-spined stickleback Apeltes quadracus , three-spined stickleback Gasterosteus aculeatus , and mummichog Fundulus heteroclitus from Sable Island ponds was confirmed using scolex morphology. The occurrence of this eel-specific parasite in these accidental hosts provides evidence for recent visits by eels Anguilla rostrata to the isolated island in the North-west Atlantic Ocean off the east coast of Canada.     

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.     

Methyltransferases do not work by compression,cratic, or desolvation effects,but by electrostatic preorganization

The enzyme catechol O‐methyltransferase (COMT) catalyzes the transfer of a methyl group from S‐adenosylmethionine to dopamine and related catechols. The search for the origin of COMT catalysis has led to different proposals and hypothesis, including the entropic, the NAC, and the compression proposals as well as the more reasonable electrostatic idea. Thus, it is important to understand the catalytic power of this enzyme and to examine the validity of different proposals and in particular the repeated recent implication of the compression idea. The corresponding analysis should be done by well‐defined physically‐based considerations that involve computations rather than circular interpretations of experimental results. Thus, we explore here the origin of the catalytic efficiency of COMT by using the empirical valence bond and the linear response approximation approaches. The results demonstrate that the catalytic effect of COMT is mainly due to electrostatic preorganization effects. It is also shown that the compression, NAC and entropic proposals do not account for the catalytic effect. Proteins 2015; 83:318–330. © 2014 Wiley Periodicals, Inc.     

Hemolysis of human erythrocytes by hypochlorous acid is modulated by amino acids,antioxidants, oxidants,membrane-perforating agents and by divalent metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA2, PLC as well as by N2, argon or by mixture of CO2 (10%) and O2 (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by Fe2+, Co2+, and V2+ and to a lesser extent with Cu2+ became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co2+ or by Mn2+. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl-induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions. Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl-generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be reconsidered.     

Stimulation of ethylene production by hydroxamic acid, in particular, by benzohydroxamic acid

The effects on the ethylene production of known inhibitors ofa cyanide-insensitive, alternative respiration in plants wereinvestigated using cotyledonary segments of cocklebur (Xanthiumpennsylvanicum Wallr.) seeds. Benzohydroxamic acid (BHAM) at3 mM stimulated ethylene production 4- to 8-fold over the control,but respiration of the segments was hardly affected at thatconcentration. The stimulatory effects of 3-chlorobenzohydroxamicacid (CLAM) and salicylhydroxamic acid were far smaller thanthat of BHAM. BHAM at 3 mM also markedly stimulated the ethyleneformation in the epicotyl or hypocotyl sections of etiolatedpea (Pisum sativum L.) and mung bean (Vigna radiata [L.] Wilczek)seedlings. Moreover, 3 mM BHAM further promoted the increasedethylene formation which was caused by administration of 1-aminocyclopro-pane-1-carboxylicacid (ACC) to the cotyledonary segments. The promoting effectsby BHAM and CLAM were also found in the conversion of ACC intoethylene in pea stem homogenates. (Received July 26, 1980; )     

Drug discovery for parasitic diseases: powered by technology,enabled by pharmacology,informed by clinical science

While prevention is a bedrock of public health, innovative therapeutics are needed to complement the armamentarium of interventions required to achieve disease control and elimination targets for neglected diseases. Extraordinary advances in drug discovery technologies have occurred over the past decades, along with accumulation of scientific knowledge and experience in pharmacological and clinical sciences that are transforming many aspects of drug R&D across disciplines. We reflect on how these advances have propelled drug discovery for parasitic infections, focusing on malaria, kinetoplastid diseases, and cryptosporidiosis. We also discuss challenges and research priorities to accelerate discovery and development of urgently needed novel antiparasitic drugs.     

Stimulation of ethylene production by hydroxamic acid, in particular, by benzohydroxamic acid

The effects on the ethylene production of known inhibitors ofa cyanide-insensitive, alternative respiration in plants wereinvestigated using cotyledonary segments of cocklebur (Xanthiumpennsylvanicum Wallr.) seeds. Benzohydroxamic acid (BHAM) at3 mM stimulated ethylene production 4- to 8-fold over the control,but respiration of the segments was hardly affected at thatconcentration. The stimulatory effects of 3-chlorobenzohydroxamicacid (CLAM) and salicylhydroxamic acid were far smaller thanthat of BHAM. BHAM at 3 mM also markedly stimulated the ethyleneformation in the epicotyl or hypocotyl sections of etiolatedpea (Pisum sativum L.) and mung bean (Vigna radiata [L.] Wilczek)seedlings. Moreover, 3 mM BHAM further promoted the increasedethylene formation which was caused by administration of 1-aminocyclopro-pane-1-carboxylicacid (ACC) to the cotyledonary segments. The promoting effectsby BHAM and CLAM were also found in the conversion of ACC intoethylene in pea stem homogenates. (Received July 26, 1980; )     

Hemolysis of Human Erythrocytes by Hypochlorous Acid is Modulated by Amino Acids,Antioxidants, Oxidants,Membrane-perforating Agents and by Divalent Metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA?, PLC as well as by N?, argon or by mixture of CO?, (10%) and 0? (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by FE²?, Co²?, and V²? and to a lesser extent with Cu²? became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co²? or by Mn²?. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl -induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions.

Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be considered.     

Graft-versus-host response,measured by splenomegaly assay,by populations of allosensitized mouse lymphocytes

The graft-vs-host (G-v-H) reactivity of sensitized or nonsensitized mouse lymphoid cell populations was measured using a splenomegaly assay. Sensitized populations were obtained either from the local lymph nodes of alloimmunized animals or from the spleens of heavily irradiated mice previously infused iv with allogeneic lymphocytes (educated cells). Immunization of animals resulted in increased G-v-H responses of the cells in their local lymph nodes. This effect was more pronounced when the immunizing cells differed only at non-H-2 transplantation antigens than when H-2-disparate strain combinations were tested. There was no evidence of a changed doseresponse profile of lymphocytes obtained from immunized mice. The G-v-H reactivity of educated cell populations was complex. The slopes of the dose-response lines obtained for lymph node cells or thymic cells educated in an H-2-disparate strain were generally lower than those obtained for nonsensitized cells. This difference was particularly evident when testing educated thymocytes. By studying the G-v-H indices obtained in A/Sn × C57B1 hybrids after inoculation of nonsensitized C57B1 lymph node cells or specifically educated C57BL lymph node cells, it was observed that the latter cells were approximately 30 times more reactive when small cell inocula were compared. On the contrary, education of lymphocytes in H-2-compatible allogeneic hosts did not result in any increment of their G-v-H reactivity. The results indicate that different methods of sensitizing lymphocyte populations against alloantigens may lead to activation of different subclasses of T-cells which differ in their mode of antigen reactivity.     

Suppression of Mitochondrial Function by Oxidatively Truncated Phospholipids Is Reversible, Aided by Bid, and Suppressed by Bcl-XL

Oxidatively truncated phospholipids are present in atherosclerotic lesions, apoptotic cells, and oxidized low density lipoproteins. Some of these lipids rapidly enter cells to induce apoptosis by the intrinsic pathway, but how such lipids initiate this process is unknown. We show the truncated phospholipid hexadecyl azelaoyl glycerophosphocholine (Az-LPAF), derived from the fragmentation of abundant sn-2 linoleoyl residues, depolarized mitochondria of intact cells. Az-LPAF also depolarized isolated mitochondria and allowed NADH loss, but did not directly interfere with complex I function. Cyclosporin A blockade of the mitochondrial permeability transition pore partially prevented the loss of electrochemical potential. Depolarization of isolated mitochondria by the truncated phospholipid was readily reversed by the addition of albumin that sequestered this lipid. Ectopic expression of the anti-apoptotic protein Bcl-X_L in HL-60 cells reduced apoptosis by the truncated phospholipid by protecting their mitochondria. Mitochondria isolated from these cells were also protected from Az-LPAF-induced depolarization. Conversely mitochondria isolated from Bid^−/− animals that lack this pro-apoptotic Bcl-2 family member were resistant to Az-LPAF depolarization. Addition of recombinant full-length Bid, which has phospholipid transfer activity, restored this sensitivity. Thus, phospholipid oxidation products physically interact with mitochondria to continually depolarize this organelle without permanent harm, and Bcl-2 family members modulate this interaction with full-length Bid acting as a co-factor for pro-apoptotic, oxidatively truncated phospholipids.Vascular cells are exposed to oxidizing radicals during normal metabolism, but especially so during physiologic and pathologic inflammatory processes. The double bonds of polyunsaturated fatty acyl residues are particularly prone to attack by radicals because the C-H bond situated between two double bonds is relatively weak, allowing a more facile abstraction of hydrogen to produce a radical (1). Because polyunsaturated fatty acyl residues are abundant and are generally esterified in the sn-2 position of the glycerol backbone, common products of oxidative attack on cells and circulating lipoproteins are phospholipids that have been peroxidized at their sn-2 position. These peroxy radicals abstract hydrogen to form hydroperoxy phospholipids, may be reduced to the corresponding alcohol, rearrange (2, 3), or fragment to generate a host of oxidatively truncated phospholipids (4–7).The shortened sn-2 residue of truncated phospholipids, which may also contain a newly introduced polar oxygen function, does not intercalate into the membrane well and is energetically favored to protrude into the aqueous phase, a conformation that disorders phospholipid packing into a bilayer (8–10). Oxidatively truncated phospholipids are more water soluble than their phospholipid precursors and readily associate with plasma albumin (11), plasma membranes (12), and even traffic into cells to lysosomes (12) or mitochondria (13) depending on the structure of the truncated phospholipid.Phospholipid oxidation products can be cytotoxic (14, 15), and at least some of these are toxic because they initiate the apoptotic process of regulated cell death (13). The manner by which oxidatively truncated phospholipids alter cell viability has been ascribed to solubilization of the plasma membrane (14), adduction of mitochondrial proteins (17), temporary physical distortion of the plasma membrane (18), or activation of acid sphingomyelinase activity that alters plasma membrane microdomains by generating ceramide (15, 19). We found that a common oxidatively truncated phospholipid, containing a 9-carbon azelaoyl fragment derived from fragmentation of sn-2 linoleoyl residues, induces apoptosis by the intrinsic caspase cascade with loss of mitochondrial function and not, apparently, from damage of the plasma membrane (13).Members of the Bcl-2 family modulate mitochondria-dependent apoptosis either by promoting apoptosis (Bid, Bad, and Bax) or obstructing this event (Bcl-2 and Bcl-X_L). Aggregation of Bax on the mitochondrial outer membrane forms ion conducting pores and Bcl-X_L associates with mitochondrial outer membranes to suppress this Bax activity (20). In contrast, Bid promotes apoptosis after cleavage to truncated Bid, a regulatory event catalyzed by activated caspase 8 (21). Bid, alone among Bcl-2 family members, displays homology to plant lipid transfer proteins and both truncated and full-length Bid will incorporate fluorescent phospholipids, and not the cognate fluorescent fatty acid, into mitochondrial membranes (22).We determined whether mitochondrial integrity or function were directly affected by oxidatively truncated phospholipids, and then whether Bcl-2 family members alter these effects as they do in other, established apoptotic signaling pathways. We find that truncated phospholipids accumulated from the extracellular environment depolarize intracellular mitochondria, that these bilayer challenged phospholipids reversibly interact with mitochondria to continually reduce their transmembrane potential, and that Bcl-2 family members modulate this interaction.     

Crop colonisation, feeding, and reproduction by the predatory beetle, Hippodamia convergens, as indicated by stable carbon isotope analysis

Abstract.  1. Habitat management to enhance natural enemy populations in agricultural systems may help regulate levels of crop pests, but little research addresses the behaviour of immigrating beneficial insects.
2. Stable carbon isotopes were used in complementary laboratory and field studies to examine colonisation behaviour of an ephemeral agricultural habitat by the lady beetle, Hippodamia convergens Guérin-Méneville.
3. Under laboratory conditions, H. convergens carbon isotope ratios, δ¹³C, changed after its food supply was shifted from a C₄- to a C₃-based diet of aphids produced on grain sorghum or cotton respectively. Final isotope ratios of adult H. convergens were closer to that of the new C₃-based diet, with most change in δ¹³C occurring within 3 days after the diet shift.
4. The carbon isotope ratios of lady beetle adults collected in cotton fields suggested that grain sorghum was a continuous source for H. convergens until many nearby sorghum fields matured and senesced.
5. When cotton aphid ( Aphis gossypii Glover) prey were absent, carbon isotope ratios of beetle populations did not change over time and virtually no egg production by H. convergens was detected. This indicates that beetles were feeding little on non-aphid resources originating in cotton.
6. With cotton aphids present, beetle isotope ratios decreased towards the carbon isotope ratio of cotton, indicating adult feeding in cotton. As a result, egg masses produced had carbon isotope ratios in the C₃ range of values.
7. The results suggest that some predator species may be retained in habitats without large prey populations, a quality essential in controlling pests in agricultural systems.