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Here we present the biological properties of the arylbipyridine platinum (II) complex (arylbipy-Pt) and describe the potential mechanism of its antitumor action which differs from that of the well-known cisplatin. Leading to the oxidative stress and lipid peroxidation, the arylbipyridine platinum (II) complex showcases the significant cytotoxicity against the glioblastoma cells as shown by the MTT test. Using the 5-ethyl-2-deoxyuridine we study the proliferative activity of glioblastoma cells to affirm that arylbipyridine platinum (II) complex does not impede cell division or DNA replication. Staining by the MitoCLox dye and 2',7'-dichlorodihydrofluorescein diacetate demonstrates that the glioblastoma cells treated with arylbipy-Pt exhibit a strong increase of the lipid peroxidation and the stimulation of the reactive oxygen species formation. The hypothesis that arylbipy-Pt promotes oxidative death of tumor cells is confirmed by control experiments using N-acetyl-L-cysteine as an antioxidant. Further evidence for the oxidative mechanism of action is provided by real-time PCR, which shows high expression levels for genes associated with the heat shock proteins HSP27 and HSP70, which can be used as markers of tumor cell ferroptosis. To elucidate the chemical nature of the arylbipy-Pt complex activity, we perform 195 Pt NMR spectroscopy and cyclic voltammetry measurements under biologically relevant conditions. The results obtained clearly indicate the structural transformation of the arylbipy-Pt complex in the DMSO-saline mixture, which is crucial for its further antitumor activity via the oxidative pathway. The found correlation between the molecular structure of arylbipy-Pt and its effect on the tumor cell cycle paves the way for the rational design of Pt complexes possessing the alternative mechanism of antitumor activity as compared to DNA intercalation, providing possible solutions to the major problems such as toxicity and drug resistance., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background/Objectives: Campylobacter jejuni (CJ) is the etiological agent of the world's most common intestinal infectious food-borne disease, ranging from mild symptoms to fatal outcomes. The development of innovative synbiotics that inhibit the adhesion and reproduction of multidrug-resistant (MDR) CJ in animals and humans, thereby preserving intestinal homeostasis, is relevant. We have created a synbiotic based on the consortium of Lactobacillus crispatus 2029 (LC2029), Ligilactobacillus salivarius 7247 (LS7247), and a mannan-rich prebiotic (Actigen ® ). The purpose of this work was to study the in vitro anti-adhesive and antagonistic activities of the created synbiotic against MDR CJ strains, along with its role in preventing intestinal barrier dysfunction, which disrupts intestinal homeostasis. Methods: A complex of microbiological, immunological, and molecular biological methods was used. The ability of the LC2029 and LS7247 consortium to promote intestinal homeostasis in vitro was assessed by the effectiveness of controlling CJ-induced TLR4 activation, secretion of pro-inflammatory cytokines, development of intestinal barrier dysfunction, and production of intestinal alkaline phosphatase (IAP). Results: All MDR CJ strains showed marked adhesion to human Caco-2, pig IPEC-J2, chicken CPCE, and bovine BPCE enterocytes. For the first time, we found that the prebiotic and cell-free culture supernatant (CFS) from the consortium of LC2029 and LS7247 strains exhibit an additive effect in inhibiting the adhesion of MDR strains of CJ to human and animal enterocytes. CFS from the LC2029 and LS7247 consortium increased the permeability of the outer and inner membranes of CJ cells, which led to extracellular leakage of ATP and provided access to the peptidoglycan of the pathogen for the peptidoglycan-degrading bacteriocins nisin and enterolysin A produced by LS7247. The LC2029 and LS7247 consortium showed a bactericidal effect on CJ strains. Co-cultivation of the consortium with CJ strains resulted in a decrease in the viability of the pathogen by 6 log. CFS from the LC2029 and LS7247 consortium prevented the growth of CJ-induced TLR4 mRNA expression in enterocytes. The LC2029 and LS7247 consortium inhibited a CJ-induced increase in IL-8 and TNF-α production in enterocytes, prevented CJ-induced intestinal barrier dysfunction, maintained the transepithelial electrical resistance of the enterocyte monolayers, and prevented an increase in intestinal paracellular permeability and zonulin secretion. CFS from the consortium stimulated IAP mRNA expression in enterocytes. The LC2029 and LS7247 consortium and the prebiotic Actigen represent a new synergistic synbiotic with anti-CJ properties that prevents intestinal barrier dysfunction and preserves intestinal homeostasis. Conclusions: These data highlight the potential of using a synergistic synbiotic as a preventive strategy for creating feed additives and functional nutrition products based on it to combat the prevalence of campylobacteriosis caused by MDR strains in animals and humans.

This paper presents the first example of the formation of acetonyl tridentate CˆNˆN complexes of arylbipyridines in the reaction of chloroplatinum complexes with acetone in the presence of alkali. The chemical structure of obtained substances was established by means of 1 H, 13 C NMR, COSY, HSQC, and HMBC techniques. The attribution of all proton and carbon signals in NMR spectra was performed using 1D and 2D NMR experiments for the synthesized acetonyl cycloplatinated complexes. A comparative analysis of the values of the C-Pt spin-spin coupling constants of the same order was carried out, which showed a significant difference in bond lengths and valence angles inthe cyclic fragments of the arylbipyridine ligand., (© 2024 John Wiley & Sons Ltd.)

Background/Objectives: The global emergence of antibiotic-resistant zooanthroponotic Escherichia coli strains, producing extended-spectrum beta-lactamases (ESBL-E) and persisting in the intestines of farm animals, has now led to the development of a pandemic of extra-intestinal infectious diseases in humans. The search for innovative probiotic microorganisms that eliminate ESBL-E from the intestines of humans and animals is relevant. Previously, we received three isolates of bifidobacteria: from milk of a calved cow (BLLT1), feces of a newborn calf (BLLT2) and feces of a three-year-old child who received fresh milk from this calved cow (BLLT3). Our goal was to evaluate the genetic identity of BLLT1, BLLT2, BLLT3 isolates using genomic DNA fingerprinting (GDF), to study the tolerance, adhesion, homeostatic and antibacterial activity of BLLT1 against ESBL-E. Methods: We used a complex of microbiological, molecular biological, and immunological methods, including next generation sequencing (NGS). Results: GDF showed that DNA fragments of BLLT2 and BLLT3 isolates were identical in number and size to DNA fragments of BLLT1. These data show for the first time the possibility of natural horizontal transmission of BLLT1 through with the milk of a calved cow into the intestines of a calf and the intestines of a child. BLLT1 was resistant to gastric and intestinal stresses and exhibited high adhesive activity to calf, pig, chicken, and human enterocytes. This indicates the unique ability of BLLT1 to inhabit the intestines of animals and humans. We are the first to show that BLLT1 has antibacterial activity against ESBL-E strains that persist in humans and animals. BLLT1 produced 145 ± 8 mM of acetic acid, which reduced the pH of the nutrient medium from 6.8 to 5.2. This had an antibacterial effect on ESBL-E. The genome of BLLT1 contains ABC-type carbohydrate transporter gene clusters responsible for the synthesis of acetic acid with its antibacterial activity against ESBL-E. BLLT1 inhibited TLR4 mRNA expression induced by ESBL-E in HT-29 enterocytes, and protected the enterocyte monolayers used in this study as a bio-model of the intestinal barrier. BLLT1 increased intestinal alkaline phosphatase (IAP) as one of the main molecular factors providing intestinal homeostasis. Conclusions: BLLT1 shows promise for the creation of innovative functional nutritional products for humans and feed additives for farm animals that will reduce the spread of ESBL-E strains in the food chain.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti- Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni , Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans , and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans -induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human β-defensin 3 in various epithelial cells. These findings support the anti- Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti- Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti- Salmonella activity, with a partial reduction upon neutralization with NaOH ( p < 0.05), suggesting the presence of anti- Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes ( p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes ( p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

Cationic copolymers based on 2-(N, N -dimethylamino)ethyl methacrylate and polyethylene glycol monomethyl ether (pDMAEMA-co-PEO) with different molecular weights have been synthesized. Their physicochemical properties were studied by NMR spectroscopy, sedimentation, and potentiometric titration. According to the data of potentiometric titration for the synthesized pegylated cationic copolymers, the apparent dissociation constants were determined in the pH range from 4.5 to 8.5. The physicochemical properties of interpolyelectrolyte complexes of these polycations with circular DNA (IPEC DNA) were also studied by dynamic light scattering, electrophoretic mobility, and TEM methods. It has been established that the diameter and electrokinetic potential (ζ-potential) of interpolyelectrolyte complexes can be varied over a wide range (from 200 nm to 1.5 μm and from -25 mV to +30 mV) by changing the ratio of oppositely charged ionizable groups in pegylated cationic copolymers and DNA, as well as by regulating medium pH. The resistance of the IPEC DNA/polycation complex to the action of nucleases was studied by electrophoresis in agarose gel; the cytotoxic effect of the polymers in vitro, and the efficiency of penetration (transfection) of IPEC DNA with PDMAEMA-co-PEO-polycations into eukaryotic cells of a cell line derived from human embryonic kidneys HEK 293 in vitro.

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens ( Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens ( Escherichia coli , Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1β, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-β, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer's patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell-adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective protein that is considered as a vaccine component for humans. LcrV mediates the delivery of Yop toxins into host cells and upregulates TLR2-dependent IL-10 production. Although LcrV can interact with the receptor-bound human interferon-γ (hIFN-γ), the significance of these interactions in plague pathogenesis is not known. In this study, we determined the parameters of specific interactions of LcrV and LcrV 68-326 with primary human thymocytes and Jurkat T-leukemia cells in the presence of receptor-bound hIFN-γ. Although the C-terminal region of hIFN-γ contains a GRRA 138-141 site needed for high-affinity binding of LcrV and LcrV 68-326 , in the hIFN-γ homodimer, these GRRA 138-141 target sites becomes accessible for targeting by LcrV or LcrV 68-326 only after immobilization of the hIFN-γ homodimer on the hIFN-γ receptors of thymocytes or Jurkat T-cells. The interaction of LcrV or LcrV 68-326 with receptor-bound hIFN-γ on the thymocytes or Jurkat T-cells caused apoptosis of both cell types, which can be completely blocked by the addition of monoclonal antibodies specific to the LEEL 32-35 and DEEI 203-206 sites of LcrV. The ability of LcrV to utilize hIFN-γ is insidious and may account in part for the severe symptoms of plague in humans., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A draft genome sequence of Lactobacillus plantarum 2025 was derived using Ion Torrent sequencing technology. The total size of the assembly (3.33 Mb) was in agreement with the genome sizes of other strains of this species. The data will assist in revealing the genes responsible for the specific properties of this strain., (Copyright © 2016 Karlyshev et al.)

In this report, we present a draft sequence of the genome of Lactobacillus rhamnosus strain 2166, a potential novel probiotic. Genome annotation and read mapping onto a reference genome of L. rhamnosus strain GG allowed for the identification of the differences and similarities in the genomic contents and gene arrangements of these strains.

This report describes a draft genome sequence of Lactobacillus crispatus 2029. The reads generated by the Ion Torrent PGM were assembled into contigs with a total size of 2.2 Mb. The data were annotated using the NCBI GenBank and RAST servers. A comparison with the reference strain revealed specific features of the genome.

This report describes a draft genome sequence of Lactobacillus plantarum 2165. The data demonstrate the presence of a large number of genes responsible for sugar metabolism and the fermentation activity of this bacterium. Different cell surface proteins, including fibronectin and mucus-binding adhesins, may contribute to the beneficial probiotic properties of this strain.

A draft genome sequence of Lactobacillus jensenii strain MD IIE-70(2) was determined using Ion PGM technology. The reads were mapped to a reference strain and assembled using a combination of tools. The genetic features revealed in this study will assist in understanding the probiotic properties of Lactobacillus bacteria.

This report describes a draft genome sequence of Lactobacillus fermentum strain 3872. The data revealed remarkable similarity to and dissimilarity with the published genome sequences of other strains of the species. The absence of and variation in structures of some adhesins and the presence of an additional adhesin may reflect adaptation of the bacterium to different host systems and may contribute to specific properties of this strain as a new probiotic.

Different common factors contribute to the antagonistic properties of Lactobacillus gasseri toward various pathogens. However, there is strain-to-strain variation in the probiotic properties of this bacterium. The draft genome sequence of L. gasseri strain 2016 determined in this study will assist in understanding the genetic basis for such variation.

The biofilm formation took place in 48 h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23.0 × 10(8) to 6.9 × 10(5) CFU/g in daily samples and to less than 10(4) CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40 % of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72 h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity.

Supernatant obtained from high dose hydrocortisone resistant thymocytes can induce migration of the bone marrow cell precursors to the periphery. This biological activity depends on the presence of the 18 kDa protein, whose amino acid sequence fits with the sequence of the secretory form of murine cyclophilin A (SP-18). Cyclophilin A isolated from the supernatant of the cortisone-resistant thymoma EL-4 shows its characteristic functional features as it demonstrates isomerase activity and binds with cyclosporine A. The cyclophilin A obtained manifests chemotactic activity that regulates migration of bone marrow cell precursors of neutrophils, T-, B- and dendritic cells.

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.

Structural and functional properties of recombinant IL-4delta2, a naturally occurring splice variant of human IL-4 with a deletion of the loop region 22-37, have been analyzed. IL-4delta2 has alpha-helical structure and most likely preserves the "up-up-down-down" topology typical of the four-helix-bundle cytokines. IL-4delta2 interacts specifically with the alpha chain of IL-4R and competes effectively with IL-4 for the common binding sites. Thus, IL-4delta2 may act as a regulator of the cytokine net, being the natural antagonist of IL-4.

Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein., (Copyright 2000 Academic Press.)

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

The cooperative structure of Fc fragments prepared from myeloma human IgG1 was studied using scanning microcalorimetry and fluorescence at pH 4.2-8.0. It was shown that the first to be melted are CH2 domains whose interaction with each other is rather weak, while that with CH3 domains is strong. Then CH3 domains which form a single cooperative block are melted. The data for the structure of the Fc fragment in solution agree with the X-ray data according to which the interaction between CH2 domains is mediated by the carbohydrate moiety while the two CH3 domains are strongly associated. The presence of intensive CH2-CH3 interaction is a distinctive feature of the state of the Fc fragment in the given pH region as compared to that at pH <4.1 [Tischenko, V. M., et al. (1982) Eur. J. Biochem. 126, 517-521; Ryazantsev, S., et al. (1990) Eur. J. Biochem. 190, 393-399]. First, cis interactions greatly increase the free energy of the native structure stabilization in CH2 domains. Second, they decrease this energy for CH3 domains when compared to the state of the latter at pH 3.8 or within the Fc' fragment (the dimer of CH3 domains). The temperature and enthalpy of melting of CH2 domains coincide in all the samples studied despite heterogeneity of the carbohydrate moiety. Thus, it may be postulated that the conservative part of CH2 domains makes a cardinal contribution to the interaction of these domains with the carbohydrate moiety.

A comparative study of the conformational and dynamics properties of the ACTH-like linear peptides, sequences of which correspond to amino acid residues 11-20 of the heavy chain of human immunoglobulin G1 Eu, residues 78-85 of human pro-interleukin-1 alpha and site 10-18 of human ACTH, was performed in aqueous solution and dimethylsulfoxide by 1H-NMR spectroscopy at 400 MHz. The peptides were shown to possess an unordered unfolded flexible conformation in aqueous solution. The revealed structural and dynamic features of the peptides are discussed together with biological activity of this class of compounds.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.

It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.

The decapeptide H2N-Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-COOH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of the human immunoglobulin G1 Eu heavy chain and displaying a 43% identity with the antigenic determinant of beta-endorphin was synthesized. Immunorphin was found to compete with 125I-beta-endorphin for high-affinity receptors on murine peritoneal macrophages (K = 2.5 +/- 0.9 x 10(-9) M) and with 3H-morphin for receptors on murine thymocytes (Ki = 2.7 +/- 0.6 x 10(-9) M) and murine macrophages (Ki = 5.9 +/- 0.7 x 10(-9) M). In particular two types of receptors to 125I-beta-endorphin with Kd1 = 6.1 +/- 0.6 x 10(-9) M and Kd2 = 3.1 +/- 0.2 x 10(-8) M were revealed on macrophages. The second type of receptors interacted with 125I-beta-endorphin, 3H-Met-enkephalin, 3H-Leu-enkephalin and 3H-morphin; the first displayed reactivity with 125I-beta-endorphin, 3H-morphin and immunorphin. The first type receptors are not present on murine brain cells nor are inhibited by naloxone. A minimum fragment of immunorphin practically completely retaining its inhibitory activity in the competition tests with 125I-beta-endorphin for common receptors on thymocytes was found to correspond to the tetrapeptide H2N-Lys-Gly-Phe-Tyr-COOH (Ki = 5.6 +/- 0.5 x 10(-9) M).

Synthesis of an artificial gene encoding the signal peptide of the Yersinia pestis capsule antigen (Caf1) was accomplished. A set of plasmids coding for hybrid proteins in which a modified sequence of the Caf1 signal peptide is connected to the amino acid sequence of the mature granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. Topography of the cleavage site of signal proteases was studied. The presence of an arginine residue within the N-terminal part of the mature human GM-CSF was shown to hinder the proper processing and translocation of the precursor through periplasmic membrane. A number of E. coli strains secreting biologically active mutants of human GM-CSF were obtained.

Understanding the interaction of Yersinia pestis with the key components of the immune system is important for elucidation of the pathogenesis of bubonic plague, one of the most severe and acute bacterial diseases. Here we report the specific, high affinity binding (Kd = 1.40 x 10(-10) M +/- 0.14 x 10(-10)) of radiolabelled human interleukin 1 beta (hIL-1 beta) to E. coli cells carrying the capsular f1 operon of Y. pestis. Caf1A outer membrane usher protein was isolated to greater than 98% purity. Competition studies with purified Caf1A, together with immunoblotting studies, identified Caf1A as the hIL-1 beta receptor. Competition between Caf1 subunit and hIL-1 beta for the same or an overlapping binding site on Caf1A was demonstrated. Relevance of these results to the pathogenesis of Y. pestis and other Gram negative bacterial pathogens with homologous outer membrane usher proteins is discussed.

A bacterial strain overproducing human prothymosin alpha was constructed based on the efficient T7 RNA polymerase transcription of human prothymosin alpha cDNA. The highest yield of the human prothymosin alpha, up to 30% of the total bacterial protein, was achieved with constructions containing 6-10 nucleotides between the Shine-Dalgarno sequence and initiation ATG codon. Unexpectedly, cells grown in the presence of inducer of T7 RNA polymerase synthesis produced substantially lower levels of prothymosin alpha than those grown in the absence of inducer. A simple procedure for prothymosin alpha isolation was elaborated, resulting in large amounts of electrophoretically pure and immunoactive protein.

The octapeptide Gly-Lys-Val-Leu-Lys-Lys-Arg-Arg (termed leukocorticotropin, LCT) corresponding to the ACTH-like sequence 81-88 of human pro-interleukin-1 alpha and its derivative Tyr-Gly-Lys-Val-Leu-Lys-Lys-Arg-Arg were synthesized. The 125I-labeled Tyr-LCT specifically interacts with one type of receptor on the surface of murine splenocytes (Kd = (1.45 +/- 0.04) x 10(-8) M, the number of binding sites is equal to 4500) and with two types of receptors on the surface of murine peritoneal macrophages (Kd1 = (5.9 +/- 1.0) x 10(-9) M and Kd2 = (2.6 +/- 2.2) x 10(-7) M). LCT and Tyr-LCT significantly increase the adenylate cyclase activity of murine peritoneal macrophages. The receptor binding and adenylate cyclase stimulation activity of LCT and Tyr-LCT are inhibited by ACTH (13-24).

125I-labelled recombinant human interferon alpha 2 (rHuIFN-alpha 2) capable of high-affinity binding (Kd = 2.46 +/- 0.18 x 10(-10) M) with receptors expressed on mouse thymocytes was obtained. Prothymosin alpha (proTM-alpha) but not cholera toxin was found to compete with radiolabelled IFN-alpha 2 for binding to the same receptor (Ki = 3.68 +/- 0.21 x 10(-11) M). The synthetic peptide covering the sequence 130-137 of IFN-alpha 2 (authors' definition: alpha-peptoferon) was shown to have the capacity to displace the labelled IFN-alpha 2 from the IFN-alpha 2/receptor complex (Ki = 7.19 +/- 0.12 x 10(-11) M). It was shown that receptors of this type are localized in plasmatic membrane fraction. Using [125I]-alpha-peptoferon, specific and saturable binding was detected on human fibroblasts and the data fitted a single binding site. Scatchard analysis yielded a Kd of 9.63 +/- 0.17 x 10(-8) M. The binding was competitively inhibited by IFN-alpha 2 (the Ki value in competition assays was 1.37 +/- 0.12 x 10(-8) M), proTM-alpha(Ki = 2.2 +/- 0.2 x 10(-7) M) and cholera toxin B subunit (Ki = 5.5 +/- 0.2 x 10(-7)). The present study has demonstrated that the sequence 130-137 of HuIFN-alpha 2 is involved in the competition of HuIFN-alpha 2, proTM-alpha and cholera toxin B subunit for common receptors on human fibroblasts.

The decapeptide Val-Lys-Lys-Pro-Gly--Ser-Ser-Val-Lys-Val (termed immunocorticotropin) corresponding to the sequence 11-20 of the variable part of the human immunoglobulin G1 heavy chain was synthesized. The ACTH-like peptide was found to have an ability to increase body temperature. Intracerebroventricular injection of 5-10 micrograms of the peptide to rabbits induced an elevation of body temperature by 0.7-1.3 degrees C. The ACTH-like peptide was shown to compete with [125I]-ACTH (13-24) for binding to receptors on murine brain synaptic membranes and to activate adenylate cyclase of these membranes.

We have cloned and sequenced the Fc-receptor-encoding gene, fcrV, from a group G streptococcus. Considerable similarity was revealed between the FcRV, FcRA76 and M proteins of group A streptococci in their signal sequences and 3' termini, and between the Fc-binding regions of FcRV and FcRA76. The promoter and terminator regions showed no homology with those of the fcrA76 and M protein-encoding genes. The A1-A4 domains of FcrV (protein V) exhibit a heptapeptide repeat motif which is characteristic of alpha-helical coiled-coil proteins. The sequence, Ser-Asn-Arg-Ala-Ala, in the outer position, 'f' of each domain is highly conserved and may be involved in FcR-IgG interactions.

A new transcription unit of the f1 gene cluster was found. The DNA sequencing revealed one long open reading frame. Deletion and frame shift mutation analyses have demonstrated the importance of a corresponding gene product for the F1 antigen biosynthesis. A homology of the deduced amino acid sequence with that of AraC family DNA-binding regulators was shown. A potential regulatory DNA region is discussed.