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PubMed2006

A cytomic approach reveals population heterogeneity of Cupriavidus necator in response to harmful phenol concentrations.

The understanding of functions of cells within microbial populations or communities is certainly needed for existing and novel cytomic approaches which grip the individual scale. Population behaviour results from single cell performances and is caused by the individual genetic pool, history, life cycle states and microenvironmental surroundings. Mimicking natural impaired environments, the paper shows that the Gram-negative Betaproteobacterium Cupriavidus necator dramatically altered its population heterogeneity in response to harmful phenol concentrations. Multiparametric flow cytometry was used to follow variations in structural cellular parameters like chromosome contents and storage materials. The functioning of these different cell types was resolved by ensuing proteomics after the cells' spatial separation by cell sorting, finding 11 proteins changed in their expression profile, among them elongation factor Tu and the trigger factor. At least one third of the individuals clearly underwent starving states; however, simultaneously these cells prepared themselves for entering the life cycle again. Using cytomics to recognise individual structure and function on the microbial scale represents an innovative technical design to describe the complexity of such systems, overcoming the disadvantage of small cell volumes and, thus, to resolve bacterial strategies to survive harmful environments by altering population heterogeneity.

Biodegradation, Environmental↗
PubMed2004

Dendritic cell-based therapies in the bench and the bedsides.

Antigen-presenting dendritic cells (DCs) represent trace population of leukocytes that are widely distributed over the whole body. DCs are regarded as inducer of antigen-specific adaptive immune responses. However, various types of functions of DCs are now exposing. In the steady-state, DCs induce immunogenic tolerance to self antigens and harmless entities and thus maintain normal homeostasis. On the other hand, in presence of non-self and dangerous entities, DCs play a cardinal role in the induction of innate immunity by producing type-1 interferons. DCs are also essential for the development of antigen-specific B-lymphocytes and plasma cells. Infection, depletion and dysfunction of DCs have been reported from patients with chronic microbial infections. Impaired functions of DCs have also been shown from patients with autoimmune diseases and allergic diseases. Patients with cancers also have phenotypic and functional impairment of DCs. These observations inspired optimism of using DC-based therapy for treating different pathological conditions. DC-based therapies showed excellent therapeutic potential in animal model of human diseases. The efficacy of DC-based therapy has not been properly evaluated in patients with different diseases. However, some clinical trials indicate that administration of antigen-pulsed DCs might be safe and possibly effective. In this review, we would first provide a description about the nature and functions of DCs that have been taught from the laboratory benches. Next, we would discuss how the information taught in the laboratory benches has been applied in patient's bedsides.

Animals↗
PubMed1998

Purification and characterisation of NADH oxidase from Thermus aquaticus YT-1 and evidence that it functions in a peroxide-reduction system.

A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT-1 by chromatography on DEAE-cellulose and AMP-Sepharose. The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit. The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH. The blue flavin semiquinone is stabilised during photo-chemical reduction of the enzyme. Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases. Reduction by the substrate NADH is incomplete, with the formation of a new long-wavelength absorption underlying the semiquinone absorption. Amino acid sequencing showed that the T aquaticus enzyme resembles other microbial flavoenzymes that function in two-enzyme systems for the reduction of peroxides, and which contain two redox-active disulphide groups in addition to the flavin. The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6-dichloroindophenol, and 5,5'dithiobis(2,2'-dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide-reducing system of Salmonella typhimurium. The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T. aquaticus NADH oxidase functions by mediating electron transfer from enzyme-bound reduced flavin to O2. The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor.

Amino Acid Sequence↗
PubMed2005

Sulfide-oxidizing activity and bacterial community structure in a fluidized bed reactor from a zero-discharge mariculture system.

In the present work we describe a comprehensive analysis of sulfide oxidation in a fluidized bed reactor (FBR) from an environmentally sustainable, zero-discharge mariculture system. The FBR received oxygen-depleted effluent from a digestion basin (DB) that is responsible for gasification of organic matter and nitrogen. The FBR is a crucial component in this recirculating system because it safeguards the fish from the toxic sulfide produced in the DB. Microscale sulfide oxidation potential and bacterial community composition within FBR biofilms were correlated to biofilter performance by integrating bulk chemical, microsensor (O2, pH, and H2S), and molecular microbial community analyses. The FBR consistently oxidized sulfide during two years of continuous operation, with an estimated average sulfide removal rate of 1.3 g of sulfide-S L(FBR)(-1) d(-1). Maximum sulfide oxidation rates within the FBR biofilms were 0.36 and 0.21 mg of sulfide-S cm(-3) h(-1) in the oxic and anoxic layers, respectively, indicating that both oxygen and nitrate serve as electron acceptors for sulfide oxidation. The estimated anoxic sulfide removal rate, as extrapolated from bench scale, autotrophic, nitrate-amended experiments, was 0.7 g of sulfide-S L(FBR)(-1) d(-1), which is approximately 50% of the total estimated sulfide removal in the FBR. Community composition analyses using denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA gene fragments from FBR samples taken at six-month intervals revealed several sequences that were closely affiliated with sulfide-oxidizing bacteria. These included the denitrifying, sulfide-oxidizing bacteria Thiomicrospira denitrificans, members of the filamentous Thiothrix genus, and sulfide-oxidizing symbionts from the Gammaproteobacteria. In addition, marine Alphaproteobacteria and Bacteroidetes species were present in all of the DGGE profiles examined. DGGE analyses showed significant shifts in the bacterial community composition between profiles over two years of sampling, indicating the presence of a diverse and dynamic microbial community within the functionally stable FBR. The FBR's combined capacity for both oxic and anoxic sulfide oxidation, as indicated by bulk chemical, microsensor, and molecular microbial analyses, gives it significant functional elasticity, which is crucial for proper performance in the dynamic environment of this mariculture system.

Aquaculture↗
PubMed2002

Phagocytosis--the mighty weapon of the silent warriors.

Professional phagocytes, comprising polymorphonuclear neutrophils and monocyte/macrophage cells, play an important role in the host defense. Any defect in their function exposes the organism to microbial intruders terminating in fatal diseases. The functional responses of the phagocytes to bacterial and fungal infections include chemotaxis, actin assembly, migration, adhesion, aggregation, phagocytosis, degranulation, and reactive oxygen species production. Superoxide generation by phagocytic NADPH oxidase is an imperative step toward bacterial killing. Phagocytes participate in inflammatory reactions and exert tumoricidal activity. They are supported by serum factors such as immunoglobulins, cytokines, complement, the acute phase reactant C-reactive protein, production of antibacterial proteins, and others. In addition to their principal task to eliminate bacteria, they are engaged in removing damaged, senescent, and apoptotic cells. Engulfed cell debris, large particles such as latex beads, fat, and oil droplets, are examples of phagocytic activity illustrated in the present review with transmission and scanning electron microscope micrographs. Numerous factors, such as diseases and stressful conditions, affect the engulfing activity of the professional phagocytes. Our experience regarding the impaired phagocytic capacity of cells in patients with diabetes and chronic renal failure is discussed. The results obtained in our laboratory from experiments detecting the effect of strenuous physical exercise, hypothermia, fasting, and abdominal photon irradiation on the phagocytic capacity of human polymorphonuclear neutrophils and rat peritoneal macrophages are hereby summarized and the reports on those subjects in the recent literature are reviewed. A variety of assays are applied for quantifying phagocytosis. Flow cytometry based on incubation of phagocytic cells with fluorescent conjugated particles and measuring the amount of fluorescence as an indicator of the engulfing capacity of the cells is a useful method. A direct visualization of the ingested particles using light or electron microscopy is a valuable tool for estimation of phagocytic function. In our hands, the use of semithin sections of embedded phagocytes following their incubation with latex particles provided satisfactory results for measuring the total number of phagocytic cells, as well as the internalizing capacity of each individual cell. Microbiological assays, the nitroblue tetrazolium test, quantitation of antibody- and antigen-mediated phagocytosis, as well as methods reviewed in detail in other reports are additional applications for determination of this intricate process.

Animals↗
PubMed1994

The Candida albicans phospholipomannan induces in vitro production of tumour necrosis factor-alpha from human and murine macrophages.

We have previously identified a Candida albicans 14,000-18,000 MW antigen reacting with anti-beta-1,2-linked oligomannosides antibodies as being a phospholipomannan (PLM). Because of the structural similarities between the C. albicans PLM and lipophosphoglycans from various microbial pathogens known to be potent tumour necrosis factor-alpha (TNF-alpha) inducers, we investigated the PLM ability to induce TNF-alpha. Incubation of human monocytic cells THP-1 with PLM led to dose-dependent production of TNF-alpha that was significantly increased by prestimulation of the cells with interferon-gamma (IFN-gamma). Production of TNF-alpha by macrophages under PLM stimulation was confirmed by using macrophages elicited from the mouse peritoneal cavity. In all investigated conditions, PLM-induced TNF-alpha production differed significantly in both kinetics and dose dependence from lipopolysaccharide (LPS) induction used as control. It appears, therefore, that the C. albicans PLM shares functional homologies with microbial lipophosphoglycans identified as pathogenicity factors, although prestimulation of the target cells was required for the PLM-derived opportunistic pathogen to trigger the cytokine network.

Animals↗
PubMed2026

Functional convergence of rTCA-related carbon-fixation potential and biochemical residue accumulation in seagrass sediments.

Seagrass meadows are globally significant blue carbon ecosystems, yet the microbial and biochemical mechanisms driving sediment organic carbon (SOC) accumulation remain poorly understood. To address this, we employed an integrated approach combining metagenomic sequencing, biochemical assays, and structural equation modeling to investigate carbon cycling in the seagrass and adjacent unvegetated sediments of Swan Lake, China. A total of 115,179 carbon fixation genes and 119,615 decomposition genes were identified, revealing distinct microbial community structures among the habitats. Seagrass sediments harbored more diverse carbon-fixing (CFMs) and decomposing microorganisms (CDMs), with 83 medium-to high-quality metagenome-assembled genomes (MAGs) recovered. While neutral community model analysis indicated that stochastic processes predominantly governed community assembly, functional analyses highlighted specific drivers of sequestration. The reductive tricarboxylic acid (rTCA) cycle emerged as the dominant carbon fixation pathway, with key genes (e.g., aclA, korA) showing strong positive correlations with SOC. Conversely, decomposition pathways for starch and lignin were negatively associated with SOC. Furthermore, seagrass sediments exhibited elevated concentrations of total amino sugars (TAS) and lignin phenols (TLP), which linked significantly to carbon fixation rather than decomposition. PLS-SEM revealed statistically significant associations among seagrass traits, environmental variables, microbial carbon-fixation potential, biochemical residue pools, and SOC, supporting a mechanistic pathway in which enhanced microbial functional potential drives the accumulation of recalcitrant biochemical residues, thereby facilitating long-term carbon retention in sediments. These findings emphasize the pivotal role of microbial anabolism and the accumulation of biosynthetic residues in sediment carbon storage, suggesting a functional convergence in seagrass-driven carbon sinks.

Metagenomics↗
PubMed2003

Shifts in archaeal communities associated with lithological and geochemical variations in subsurface Cretaceous rock.

Subsurface microbial community structure in relation to geochemical gradients and lithology was investigated using a combination of molecular phylogenetic and geochemical analyses. Discreet groundwater and substratum samples were obtained from depths ranging from 182 to 190 m beneath the surface at approximately 10-cm intervals using a multilevel sampler (MLS) that straddled Cretaceous shale and sandstone formations at a site in the southern San Juan Basin in New Mexico. DNA and RNA were extracted directly from quartzite sand substratum loaded into individual cells of the MLS and colonized in situ. Polymerase chain reaction (PCR)-mediated T-RFLP analysis of archaeal rRNA genes (rDNA) in conjunction with partial sequencing analysis of archaeal rDNA libraries and quantitative RNA hybridization with oligonucleotide probes were used to probe community structure and function. Although total microbial populations remained relatively constant over the entire depth interval sampled, significant shifts in archaeal populations, predominantly methanogens, were observed. These shifts coincided with the geochemical transition from relatively high methane (26 mM), low sulphate (< 3 mg l(-1)) conditions in the region adjacent to the organic matter-rich shale to relatively low-methane (< 0.5 mM), high-sulphate (48 mg l(-1)) conditions in the organic-poor sandstone beneath the shale. These results indicated that active, phylogenetically diverse archaeal communities were present in the subsurface Cretaceous rock environment at this site and that major archaeal clades shifted dramatically over scales of tens of centimetres, corresponding to changes in the lithology and geochemical gradients.

Archaea↗
PubMed2002

Positive and negative regulation of pathogen induced dendritic cell function by G-protein coupled receptors.

The induction of IL-12 from dendritic cells (DC) is a major initiating step in host resistance to intracellular pathogens. We have studied the regulation of this response using an in vivo model in which IL-12 production by splenic CD8alpha+ DC is followed after injection of a soluble extract (STAg) of the protozoan parasite Toxoplasma gondii. Our findings indicate that the potent IL-12 response observed is highly dependent on both the chemokine receptor CCR5 and G(i)-protein coupled signaling. In addition, we have examined the basis of the unresponsiveness of DC to secondary STAg injection which occurs following primary exposure to this parasite stimulus. Our results demonstrate that this refractory state correlates with the down-regulation of CCR5 expression on DC which, in turn, appears to depend on the induction of endogenous lipoxin A(4) (LXA(4)), a product of arachidonic acid metabolism. Since LXA(4) is known to also signal through a G-protein coupled receptor pathway, these findings taken together support a major role for G-protein signaling in the regulation of microbial-induced DC function.

Animals↗
PubMed2006

Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach.

Escherichia coli is a model laboratory bacterium, a species that is widely distributed in the environment, as well as a mutualist and pathogen in its human hosts. As such, E. coli represents an attractive organism to study how environment impacts microbial genome structure and function. Uropathogenic E. coli (UPEC) must adapt to life in several microbial communities in the human body, and has a complex life cycle in the bladder when it causes acute or recurrent urinary tract infection (UTI). Several studies designed to identify virulence factors have focused on genes that are uniquely represented in UPEC strains, whereas the role of genes that are common to all E. coli has received much less attention. Here we describe the complete 5,065,741-bp genome sequence of a UPEC strain recovered from a patient with an acute bladder infection and compare it with six other finished E. coli genome sequences. We searched 3,470 ortholog sets for genes that are under positive selection only in UPEC strains. Our maximum likelihood-based analysis yielded 29 genes involved in various aspects of cell surface structure, DNA metabolism, nutrient acquisition, and UTI. These results were validated by resequencing a subset of the 29 genes in a panel of 50 urinary, periurethral, and rectal E. coli isolates from patients with UTI. These studies outline a computational approach that may be broadly applicable for studying strain-specific adaptation and pathogenesis in other bacteria.

Chromosomes, Bacterial↗
PubMed2004

Raman microscopic analysis of single microbial cells.

We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species. In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species. The use of multivariate methods subsequently allowed taxa discrimination. Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination. Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate. A single strain was grown in media containing incrementally varying ratios of (13)C(6) to (12)C(6) glucose, and it was found that (13)C incorporation shifted characteristic peaks to lower wavenumbers. These findings suggest that Raman microscopy has significant potential for studies requiring the taxonomic identity and functioning of single microbial cells to be determined.

Bacteria↗
PubMed2026

Promises and pitfalls of long-read sequencing for resolving microbial complexity.

Long-read sequencing (LRS) has driven a transition in microbial genomics, overcoming the assembly fragmentation inherent to short-read sequencing. This review elucidates the impact of LRS across isolate genomics, metagenomics, and multi-omics domains. By spanning extensive repetitive regions, LRS facilitates the reconstruction of circular chromosomes and precisely resolves mobile genetic elements (MGEs). In metagenomics, LRS enables strain-level resolution, the recovery of circular metagenome-assembled genomes, and the precise localization of MGEs within host replicons. Furthermore, the single-molecule, amplification-free properties of LRS provide enhanced resolution of native epigenetic modifications and full-length transcriptomes. Despite these advancements, widespread implementation remains constrained by multidimensional challenges, including stringent high-molecular-weight DNA requirements, depth deficits, and computational overhead. Nevertheless, LRS is increasingly becoming the method of choice for isolate genomics and metagenomics. As detection technologies and algorithms progress, LRS will further improve our ability to decipher the structural and functional diversity of microbial ecosystems.

Metagenomics↗
PubMed1987

Clarification and quantitation of primary (tissue) and secondary (microbial) catabolites of riboflavin that are excreted in mammalian (rat) urine.

Riboflavin derivatives were quantitated and identified in urine of rats fed 0, 2 and 6 micrograms riboflavin/g diet per day both with and without added succinyl sulfathiazole for 6 wk. Two rats from each dietary group were placed in metabolic cages and urine was collected in the dark for 24 h. On the fourth week, a third animal from each group received an i.p. injection of [2-14C]riboflavin before being placed in a metabolic cage and urine collected in the dark for 48 h. Urine samples were extracted with phenol for flavin components and with chloroform for lumichrome and derivatives. Riboflavin was the predominant flavin excreted by rats in all dietary groups, followed by hydroxymethylriboflavins and smaller amounts of flavin mononucleotide (FMN), lumiflavin and 10-hydroxyethylflavin. Carboxylumichromes accounted for 5-10% of the total flavin-derived fluorescence in urine of rats fed 2 and 6 micrograms riboflavin/g diet and were reduced to approximately 3% when sulfathiazole was added to the base diets. Carboxylumichromes were absent from urine of riboflavin-deficient rats. Riboflavin accounted for 85-90% of the recovered radioactivity of all radioactive urine extracts; no radioactively labeled carboxylumichromes were detected. These results indicate that hydroxymethylriboflavins are primary catabolites of riboflavin derived from tissue microsomal oxidations, whereas carboxylumichromes reflect the continued oxidation of ring hydroxymethyl functions plus gut microbial cleavage of the side chain of flavin.

Animals↗
PubMed1999

Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere.

The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities.

Biodegradation, Environmental↗
PubMed1998

In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members.

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of an Acinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and the meta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in P. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the Pu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pm promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of Pm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

Acinetobacter↗
PubMed2002

[Identification of CD11c+ myeloid dentritic cells in adenoids and in nasal mucosa of patients with and without allergies].

BACKGROUND: Dendritic cells form a link between innate and acquired immunity. They are capable to detect pathogens based on the recognition of pathogen-associated microbial molecules and trigger the appropriate type of immune responses. In humans, three major subsets of dendritic cells can be distinguished, Langerhans cells of the skin, myeloid DC (MDC) and plasmacytoid DC (PDC). It was reported that PDC infiltrate nasal mucosa in allergen-induced rhinitis. Information about the role of MDC in nasal mucosa and the corresponding mucosa-associated lymphoid tissue, the nasopharyngeal adenoids, is limited. PATIENTS AND METHODS: : Here we examined the presence of MDC in adenoids and in nasal mucosa of healthy individuals (n = 9) and in patients with allergic rhinitis. MDC were detected by flow cytometry by positive staining for MHC II and CD11c and the lack of lineage markers. Dead cells were excluded from analysis. RESULTS: In adenoids, 0.4 % of all cells were MDC. Considerable numbers of MDC could also be detected in nasal mucosa. No difference was found between healthy individuals and patients with allergies (0.3 % vs. 0.45 % MDC; p = 0.12). Interestingly, MDC were absent in patients who received treatment with glucocorticoids, while very high numbers of MDC were found in patients who recently had upper respiratory tract infections. CONCLUSION: Our results demonstrate for the first time the presence of MDC in nasal mucosa. MDC numbers were similar in healthy individuals and in patients with allergy. This study forms the basis for examining the role of MDC in the pathogenesis of allergic rhinitis, and for the modulation of MDC functional activity with microbial molecules such as CpG oligonucleotides.

Adenoids↗
PubMed2006

Antimicrobial barrier of an in vitro oral epithelial model.

OBJECTIVE: Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture. DESIGN: The tissue model was characterized for keratin and beta-defensin expression. Altered expression of beta-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-alpha in the medium. These were compared to the response in traditional submerged oral epithelial cell culture. RESULTS: The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3-600-fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100-fold in response to TNF-alpha in the tissue model and 50-fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression. CONCLUSIONS: The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier.

Cells, Cultured↗
PubMedDate not supplied

[Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria].

We conducted a comparative study of the effects of alpha-amino-gamma-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of gram-positive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10(-5)-10(-3) M HSL or 10(-5)-10(-4) M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10(-4)-10(-3) M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10(-4) to (1.5-2.5) 10(-4) M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10(-4) M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075-0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions on the microbial community level.

4-Butyrolactone↗