Human lysosomal genes: Arylsulfatase A and β-Galactosidase

The segregation of human lysosomal arylsulfatase A (ARS-A) has been evaluated in 50 primary hybrid clones derived from four separate fusions involving WBCs from two unrelated individuals and three hamster cell lines. ARS-A was expressed in the hybrids as a dimeric molecule of very similar or identical subunits. The expression of this enzyme was concordant with that of mitochondrial aconitase (ACON-M), an isozyme assigned to chromosome 22, in all 50 clones and with chromosome 22 segregation in all but one of the 29 karyotyped hybrids. No other human chromosome cosegregated with 22 in these clones, suggesting that this enzyme is specified in hybrid cells by a locus (or loci) on a single chromosome. beta-Galactosidase (B-GAL) expression was analyzed with two different electrophoresis systems and with a number of cell extract preparation methods in 39 of the primary hybrid clones. The B-GAL isozyme expressed in these hybrid cells was concordant with the expression of glutathione peroxidase-1 (GPX-1), an isozyme assigned to chromosome 3, in all 39 clones and with the segregation of this chromosome in 97% of the 29 karyotyped hybrids. These observations substantiate the prior tentative assignments of an ARS-A locus to chromosome 22 and a B-GAL locus to chromosome 3 (Bruns et al., 1978a, b). The implications of the chromosome assignments of loci for 12 human lysosomal enzymes for the cellular assembly of these organelles are discussed.     

Chimeric analogs of human β-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human β-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.     

Hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza A virus

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.     

Kinetics of Avibactam Inhibition against Class A,C, and D β-Lactamases

Avibactam is a non-β-lactam β-lactamase inhibitor with a spectrum of activity that includes β-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k₂/K_i) varied across the enzyme spectrum, from 1.1 × 10¹ m⁻¹s⁻¹ for OXA-10 to 1.0 × 10⁵ for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.     

Reduced tonsillar expression of human β-defensin 1, 2 and 3 in allergic rhinitis

Airway infections are known to cause exacerbations of allergy and asthma. Tonsils constitute a primary site for microbial recognition and triggering of the immune system in the airways. Human β-defensins (HBDs) are antimicrobial peptides with an important role in this defense. Our aim was to investigate HBD1-3 in tonsillar tissue and their potential role in allergic rhinitis (AR). Tonsils, obtained from patients with AR and non-allergic controls, and isolated tonsillar CD4(+), CD8(+) and CD19(+) lymphocytes were analyzed for HBD1-3 expression using real-time RT-PCR and/or immunohistochemistry. Tonsillar tissue, mixed tonsillar lymphocytes and airway epithelial cells (AECs) were cultured with or without IL-4, IL-5, IL-13 or histamine followed by measurements of HBD1-3 release using ELISA. HBD1-3 were present in tonsillar tissue, including epithelial, CD4(+), CD8(+) and CD19(+) cells. The expression was reduced in allergic compared to healthy tonsils. Stimulation of AECs with IL-4, IL-5 and histamine down-regulated the HBD release, whereas no effects were seen in cultured tonsils or lymphocytes. This study demonstrates presence of HBD1-3 in tonsils and that the levels are reduced in patients with AR. Together with the down-regulation of HBDs in epithelial cells in the presence of allergic mediators suggest that AR patients have an impaired antimicrobial defense that might make them more susceptible to respiratory tract infections.     

The human solute carrier family 11 member 1 protein (SLC11A1): linking infections, autoimmunity and cancer?

SLC11A1 is known to link infections, autoimmunity and cancers. A review is presented of the mechanisms by which a balance is maintained between infections caused by pathogens (viral, bacterial and protozoan; intracellular and extracellular) and disorders resulting from (acute or chronic) inflammation, and of the interactions that determine how the initial innate immune system directs subsequent acquired immune responses in human populations.     

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that -defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine -defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with -defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel -defensin genes share the typical exon–intron structure and are highly homologous to published bovine -defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several -defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of -defensins from literature, the abundance of -defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that -defensins may play a role in local host defense during udder infections.The nucleotide sequence data reported in this article have been submitted to EMBL and have been assigned the accession numbers AJ563279–AJ563283, AJ567353–AJ567365, AJ567990–AJ567993, and AJ620296.     

Human β-defensin 3 suppresses Porphyromonas gingivalis lipopolysaccharide-induced inflammation in RAW 264.7 cells and aortas of ApoE-deficient mice

Human beta-defensin 3 (hBD3) is an antimicrobial peptide showing immunomodulatory effect on both innate and acquired immune response. Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the vascular wall. In this study, we evaluated whether hBD3 could attenuate the atherosclerosis development accelerated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) with apolipoprotein E-deficient (ApoE^−/−) mice. We observed that, in vivo, hBD3 inhibited serum MCP-1, sICAM-1 levels of ApoE-deficient mice exposed to Pg-LPS in a chronic inflammation model. Serum levels of total cholesterol (TC) and low-density lipoprotein (LDL) were also markedly reduced with hBD3 intervention. In addition, thinned vascular walls, less macrophage infiltration and the formation of atherosclerotic lesions were observed in the hBD3-treated group. Furthermore, in vitro, hBD3 profoundly suppressed the production of TNF-α and IL-6 in RAW 264.7 cells induced by Pg-LPS in a dose-dependent manner. Moreover, hBD3 attenuated the phosphorylation of p38 and ERK1/2 in the mitogen-activated protein kinase (MAPK) pathway. Taken together, our work has revealed that hBD3 exhibits potent anti-inflammatory properties both in vitro and in vivo, and this effect might be correlated with inhibition of MAPK pathway.     

Farnesol promotes epithelial cell defense against Candida albicans through Toll-like receptor 2 expression,interleukin-6 and human β-defensin 2 production

Farnesol, a quorum-sensing molecule, regulates virulence and morphogenesis in Candida albicans and is involved in various human pathologies including oral candidiasis. Oral epithelial cells are involved in innate immunity against Candida infections via Toll-like receptors (TLRs) and inflammatory mediators. We investigated the effects of farnesol on host cells and its possible synergistic interaction with gingival epithelial cells against C. albicans infection by studying the expression of TLR2, 4 and 6. The production of IL-6, IL-8, and human β-defensins 1 and 2 was also examined using engineered human oral mucosa tissue put in contact with various concentrations of farnesol with and without C. albicans. Our findings indicate that 24 h after contact with C. albicans, epithelial cells expressed more TLR2 than did non-infected cells. The addition of exogenous farnesol upregulated the TLR2 expression by the gingival epithelial cells in the presence or absence of C. albicans. In contrast, TLR4 was down regulated when farnesol was added to the tissue with or without C. albicans. Finally, farnesol alone was shown to have no effect on TLR6, yet in the presence of both C. albicans and farnesol, TLR6 expression was down regulated. Farnesol modulated TLR2 expression by the epithelial cells following tissue contact with C. albicans. This effect was paralleled by IL-6 but not IL-8 secretion. Farnesol’s effect on innate immunity was strengthened by its capacity to increase human β-defensin 2 production, and by the efficacy of β-defensin against C. albicans growth. Overall results showed that exogenous farnesol promoted epithelial cell defense against C. albicans infection through the involvement of TLR2, IL-6, and human β-defensin 2.     

Avian β-defensin variation in bottlenecked populations: the Seychelles warbler and other congeners

β-defensins are important components of the vertebrate innate immune system responsible for encoding a variety of anti-microbial peptides. Pathogen-mediated selection is thought to act on immune genes and potentially maintain allelic variation in the face of genetic drift. The Seychelles warbler, Acrocephalus sechellensis, is an endemic passerine that underwent a recent bottleneck in its last remaining population, resulting in a considerable reduction in genome-wide variation. We genotyped avian β-defensin (AvBD) genes in contemporary (2000–2008) and museum samples (1876–1940) of the Seychelles warbler to investigate whether immunogenetic variation was lost through this bottleneck, and examined AvBD variation across four other Acrocephalus species with varying demographic histories. No variation was detected at four of the six AvBD loci screened in the post-bottleneck population of Seychelles warbler, but two silent nucleotide polymorphisms were identified at AvBD8 and one potentially functional amino-acid variation was observed at AvBD11. Variation in the Seychelles warbler was significantly lower than in the mainland migratory congeneric species investigated, but it similar to that found in other bottlenecked species. In addition, screening AvBD7 in 15 museum specimens of Seychelles warblers sampled prior to the bottleneck (1877–1905) revealed that this locus possessed two alleles previously, compared to the single allele in the contemporary population. Overall, the results show that little AvBD variation remains in the Seychelles warbler, probably as a result of having low AvBD diversity historically rather than the loss of variation due to drift associated with past demographic history. Given the limited pathogen fauna, this lack of variation at the AvBD loci may currently not pose a problem for this isolate population of Seychelles warblers, but it may be detrimental to the species’ long-term survival if new pathogens reach the population in the future.     

A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

Identification and characterization of a novel antibacterial peptide,avian β-defensin 2 from ducks

In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, −20°C to 100°C) and pH values (range, 3 to 12)     

CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene–gene interactions

Gene–environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene–gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene–gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene–gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9–8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5–15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.     

N-Propargylic β-enaminones in breast cancer cells: Cytotoxicity,apoptosis, and cell cycle analyses

Breast cancer is one of the most common cancers worldwide and the discovery of new cytotoxic agents is needed. Enaminones are regarded to be a significant structural motif that is found in a variety of pharmacologically active compounds however the number of studies investigating the anticancer activities of N-propargylic β-enaminones (NPEs) is limited. Herein we investigated the potential cytotoxic and apoptotic effects of 23 different NPEs (1-23) on human breast cancer cells. Cytotoxicity was evaluated via MTT assay. Apoptotic cell death and cell cycle distributions were investigated by flow cytometry. CM-H2DCFDA dye was used to evaluate cellular ROS levels. Expression levels of Bcl-2, Bax, p21, and Cyclin D1 were measured by quantitative real-time PCR. ADME properties were calculated using the ADMET 2.0 tool. NPEs 4, 9, 16, and 21 showed selective cytotoxic activity against breast cancer cells with SI values >2. NPEs induced apoptosis and caused significant changes in Bcl-2 and Bax mRNA levels. The cell cycle was arrested at the G0/G1 phase and levels of p21 and Cyclin D1 were upregulated in both breast cancer cells. ROS levels were significantly increased by NPEs, suggesting that the cytotoxic and apoptotic effects of NPEs were mediated by ROS. ADME analysis revealed that NPEs showed favorable distributions in both breast cancer cell lines, meaning good lipophilicity values, low unfractionated values, and high bioavailability. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.