Chemodiversity Associated with Cytotoxicity and Antimicrobial Activity of Piper aduncum var. ossanum

Chemical analysis, antimicrobial activity and cytotoxic effects of essential oils (EOs) from leaves of Piper aduncum var. ossanum from two localities Bauta (EO‐B) and Ceiba (EO‐C), Artemisa Province, Cuba, were determined. EOs were obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry. EO‐B demonstrated higher activity against S. aureus and L. amazonensis; while a lower cytotoxicity on mammalian cells was observed. Both EOs displayed the same activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania infantum. Both EOs were inactive against Escherichia coli and Candida albicans.     

重组创伤弧菌溶细胞素缬氨酸V201和V289突变对其活性的影响

将重组创伤弧菌溶细胞素A（recombinant Vibrio vulnificus hemolysin,rVvhA）第201位和第289位的缬氨酸定点突变为甘氨酸,并表达rVvhAval201gly,val289gly突变蛋白。检测突变蛋白与未突变蛋白的溶血活性、对胞内钙离子浓度、钾离子外流及对细胞损伤的影响。结果显示,与rVvhAval201gly,val289gly的溶血活性和细胞毒性均降低,诱导人类脐静脉内皮细胞（human umbilical veinendothelial cells,HUVEC）凋亡、胞内钙离子内流和钾离子外流等作用均受到抑制。实验结果表明,在创伤弧菌溶细胞素的活性发挥中,rVvhA的V201和V289两个氨基酸与该蛋白质损伤靶细胞时引起胞内外离子平衡失调有关,并能影响该蛋白质的溶血活性和细胞毒作用。     

组氨酸残基在乙醇酸氧化酶活性中的作用

DEPC能显著抑制GAO的活性。其失活速度表现为假一级动力学特性,并和抑制剂浓度成线性正比关系。底物乙醇酸可保护GAO免受DEPC抑制,羟胺能使被抑制的酶重新复活。光谱测定表明,被抑制的酶只有组氨酸残基被修饰,而酪氨酸残基未被修饰,修饰前后酶的氨基含量均无变化。反应动力学表明,在35℃下,GAO中有一个pK为6.5的解离基团和催化活性有关,其解离⊿H为31610 J/mol。因此组氨酸残基为GAO催化活性的一个必需基团。     

Catalytic Function of the Residues of Phenylalanine and Tyrosine Conserved in Squalene-Hopene Cyclases

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-π interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three redsidues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Tyrosine Residues at the Carboxyl Terminus of Vav1 Play an Important Role in Regulation of Its Biological Activity

The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-γ associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.     

Isolation of Sugar Compounds of Asparagine,Phenylalanine, Tyrosine and Valine from Cured Tobacco Leaves

Scanning electron microscopy and rigidity characterization were used in this study of myosin gelation in the presence of actin. The heat-induced gel formation of myosin was aided by the addition of actin to myosin at a molar ratio of 1: 2.7. However, this actin effect was not observed when the actin-binding site(s) of myosin was blocked by p-chloromercuri-benzoate treatment or denaturation, or when the myosin-binding site(s) of actin was blocked by trinitrophenylation.

The effect also disappeared with an aging myosin-actin mixture, with ATP or pyrophosphate as dissociating agent prior to thermal treatment of the system.

The present findings indicate the indispensability of myosin binding to actin for enhancing the thermal gelation of myosin. The gelation process appears to involve a large electrostatic contribution, as well as such chemical reactions as oxidation of SH groups.     

Synthetic Antimicrobial Peptides. II. Antimicrobial and Hemolytic Activity of Cationic Peptides Containing Cysteine Residues with Free Sulfhydryl Groups

Russian Journal of Bioorganic Chemistry - The antimicrobial and hemolytic activities of R9F2С2 (P1ss), (KFF)3KС2 (P2ss), and (RAhaR)4AhaβAС2 (P3ss) (where Aha is...     

Elevated Expression of the Serine-Arginine Protein Kinase 1 Gene in Ovarian Cancer and Its Role in Cisplatin Cytotoxicity In Vitro

Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. In the present study we have tested whether serine-arginine protein kinase 1 (SRPK1), a major regulator of splicing factors, is involved in ovarian cancer progression and plays a role in chemo-sensitivity. By Western blot analyses, SRPK1 protein was found to be overexpressed in 4 out of 6 ovarian cancer cell lines as compared with an immortalized ovarian surface epithelial cell line; and in 55% of ovarian tumor samples as compared with non-neoplastic ovarian tissue samples. Reduction of SRPK1 expression using small interfering RNA (siRNA) encoding small hairpin RNA in ovarian cancer cells led to (i) reduced cell proliferation rate, slower cell cycle progression and compromised anchorage-independent growth and migration ability in vitro, (ii) decreased level of phosphorylation of multiple serine-arginine proteins, and P44/42MAPK and AKT proteins, and (iii) enhanced sensitivity to cisplatin. Together, these results suggest that elevated SRPK1 expression may play a role in ovarian tumorigenesis and SRPK1 may be a potential target for ovarian cancer therapy.     

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. “Nauty”) root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO₂ partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.     

Key Role of Disulfide Bridges in the Antimicrobial Activity of Beta-Defensin from Olive Flounder

International Journal of Peptide Research and Therapeutics - Proteins that contain multiple disulfide bonds (SS bonds) expressed in Escherichia coli are usually problematic. This study reports the...     

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A₂, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A₁ and A₂ activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A₂ activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.     

Role of Phenylalanine Deaminase and Tyrase in the Lignification of Bamboo

A pronounced increase of the activity of phenylalanine deaminase and tyrase during the lignification of bamboo shoots was observed. With progressing maturation from the basal part to the upper part of immature bamboos the pattern of the activity of the enzymes moved toward the upper part of the tissues, where the most active lignification was taking place, and the amount of cinnamic acid derivatives in the tissues was found to be in good accordance with the activity of the enzymes. l-Phenylalanine-G-¹⁴C and l-tyrosine-G-¹⁴C were both well incorporated into the lignin of bamboos. These results indicate that phenylalanine deaminase and tyrase are synthesized progressively just before the lignification of the bamboos and by the enzymes l-phenylalanine and l-tyrosine are deaminated to cinnamic acid derivatives and incorporated into lignin.     

Regulation of C-type Lectin Antimicrobial Activity by a Flexible N-terminal Prosegment

Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity.The gastrointestinal tracts of mammals are heavily colonized with vast symbiotic microbial communities and are also a major portal of entry for bacterial pathogens. To cope with these complex microbial challenges, intestinal epithelial cells produce a diverse repertoire of protein antibiotics from multiple distinct protein families (1). These proteins are secreted apically into the luminal environment of the intestine where they play a pivotal role in protecting against enteric infections (2, 3) and may also function to limit opportunistic invasion by symbiotic bacteria (4).We previously identified lectins as a novel class of secreted antibacterial proteins in the mammalian intestine. RegIIIγ is a member of the RegIII subgroup of the C-type lectin family and is expressed in the small intestine in response to microbial cues (5), stored in epithelial cell secretory granules, and released into the small intestinal lumen (5). Similarly, HIP/PAP (hepatointestinal pancreatic/pancreatitis-associated protein; the human ortholog of RegIIIγ)⁶ is expressed in the human intestine (6) and is up-regulated in patients with inflammatory bowel disease (7). These proteins are produced in multiple epithelial lineages, including enterocytes and Paneth cells (5, 6). Both RegIIIγ and HIP/PAP are directly bactericidal at low micromolar concentrations for Gram-positive bacteria (5), revealing a previously unappreciated biological function for mammalian lectins. The antibacterial functions of RegIIIγ and HIP/PAP are dependent upon binding bacterial targets through interactions with peptidoglycan (5). As peptidoglycan is localized on surfaces of Gram-positive bacteria but is buried in the periplasmic space of Gram-negative bacteria, this binding activity provides a molecular explanation for the Gram-positive specific bactericidal effects of these lectins. Although the mechanism of lectin-mediated antibacterial activity remains unclear, RegIIIγ and HIP/PAP have been shown to elicit extensive damage to the cell surfaces of targeted bacteria (5).In this study, we show that C-type lectin bactericidal activity is under stringent post-translational control. RegIIIγ and HIP/PAP each undergo in vivo proteolytic removal of a flexible anionic N-terminal prosegment that maintains the proteins in a biologically inactive state. NMR spectroscopy suggests that the prosegment functions by controlling a two-state conformational switch between the biologically active and inactive states of the protein. We propose that this regulatory mechanism allows the host to restrict expression of RegIII lectin antibacterial activity to the intestinal lumen. Together, our findings represent a unique example of post-translational control of C-type lectin biological activity, and provide novel insight into the regulation of lectin-mediated innate immunity in the mammalian intestine.     

Role of Glycine in the N-Methyl-d-Aspartate-Mediated Neuronal Cytotoxicity

Current evidence indicates that glutamate acting via the N-methyl-D-aspartate (NMDA) receptor/ion channel complex plays a major role in the neuronal degeneration associated with a variety of neurological disorders. In this report the role of glycine in NMDA neurotoxicity was examined. We demonstrate that NMDA-mediated neurotoxicity is markedly potentiated by glycine and other amino acids, e.g., D-serine. Putative glycine antagonists HA-966 and 7-chlorokynurenic acid were highly effective in preventing NMDA neurotoxicity, even in the absence of added glycine. The neuroprotective action of HA-966 and 7-chlorokynurenic acid, but not that of NMDA antagonists 3-(2-carboxypiperazine-4-yl)propylphosphonate and MK-801, could be reversed by glycine. These results indicate that glycine, operating through a strychinine-insensitive glycine site, plays a central permissive role in NMDA-mediated neurotoxicity.     

APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)⁵ protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants.