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B Henderson

Publications and source records attributed to B Henderson.

At least 37 records · Page 2Linked to original sources
PubMed2001

Identification of photolabile outer membrane proteins of Porphyromonas gingivalis.

As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections need to be developed. This is particularly pertinent with respect to the periodontal diseases--the most common chronic bacterial infections of man. The use of a photosensitizer in combination with red light has been demonstrated to be effective in killing several human pathogens, including the oral bacterium, Porphyromonas gingivalis, a major pathogen in periodontitis. Killing was associated with alterations in the molecular masses of several outer membrane and plasma membrane proteins and these may be therapeutic targets for photodynamic therapy and other antimicrobial approaches. To identify these photolabile proteins, we have used a panel of monoclonal antibodies raised to whole P. gingivalis. A number of the antibodies recognized various photolabile proteins. Using a combination of Western blotting and protein sequencing the predominant photolabile proteins in P. gingivalis have been identified as the major secreted/cell surface proteases--Lys and Arg gingipain.

Adhesins, Bacterial↗
PubMed2001

Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

Adipocytes↗
PubMed2001

Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein.

Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin beta1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 -10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.

Animals↗
PubMed2001

Cloning and expression of the Actinobacillus actinomycetemcomitans thioredoxin (trx) gene and assessment of cytokine inhibitory activity.

Thioredoxin is a ubiquitous redox control and cell stress protein. Unexpectedly, in recent years, thioredoxins have been found to exhibit both cytokine and chemokine activities, and there is increasing evidence that this class of protein plays a role in the pathogenesis of inflammatory diseases. In spite of this evidence, it has been reported that the oral bacterium and periodontopathogen Actinobacillus actinomycetemcomitans secretes an immunosuppressive factor (termed suppressive factor 1 [SF1] [T. Kurita-Ochiai and K. Ochiai, Infect. Immun. 64:50-54, 1996]) whose N-terminal sequence, we have determined, identifies it as thioredoxin. We have cloned and expressed the gene encoding the thioredoxin of A. actinomycetemcomitans and have purified the protein to homogeneity. The A. actinomycetemcomitans trx gene has 52 and 76% identities, respectively, to the trx genes of Escherichia coli and Haemophilus influenzae. Enzymatic analysis revealed that the recombinant protein had the expected redox activity. When the recombinant thioredoxin was tested for its capacity to inhibit the production of cytokines by human peripheral blood mononuclear cells, it showed no significant inhibitory capacity. We therefore conclude that the thioredoxin of A. actinomycetemcomitans does not act as an immunosuppressive factor, at least with human leukocytes in cultures, and that the identity of SF1 remains to be elucidated.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Mycobacterium tuberculosis chaperonin 60.1 is a more potent cytokine stimulator than chaperonin 60.2 (Hsp 65) and contains a CD14-binding domain.

Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed both M. tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2. This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins. The M. tuberculosis Cpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies. The cytokine-inducing activity of both chaperonins is extremely resistant to heat. Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.

Amino Acid Sequence↗
PubMed2001

Identification of the exported proteins of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans by using alkaline phosphatase fusions.

A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria.

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

Adhesins, Bacterial↗
PubMed2001

Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis.

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.

Actinobacillus Infections↗
PubMed2001

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis.

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.

Acid-Base Equilibrium↗
PubMed2001

Pro-inflammatory--anti-inflammatory cytokine dynamics mediated by cytokine-receptor dynamics in monocytes.

Many of the major human diseases, both infectious (septic shock syndromes) and idiopathic (for example, rheumatoid arthritis), are driven by the production of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) produced by monocytes and macrophages. These key pro-inflammatory cytokines can, in turn, stimulate the production of additional cytokines which, in totality, generate tissue pathology. A major deactivator of activated, cytokine-producing monocytes and macrophages is the anti-inflammatory cytokine interleukin-10 (IL-10). It is known that the interactions between these three cytokines are pivotal in terms of health and pathology, but almost nothing is known of the dynamics of these interactions. In this study we have modelled the autocrine interactions of TNF-alpha, IL-1 and IL-10 with monocytes. The model constructed is a six-dimensional, continuous-time dynamical system, with free IL-1 and IL- 10 concentrations in the cell's vicinity, and the proportions of bound and free IL-1 and IL-10 cell-surface receptors, which transduce the cell's response to stimulation, as the state variables. The monocyte is assumed to be initially in a quiescent state, and it is stimulated to produce IL-1 by an external stimulus (e.g. exposure to TNF-alpha or lipopolysaccharide, LPS). This in turn invokes an autocrine IL-1 response, and also induces the production of the anti-inflammatory cytokine IL-10, which acts to downregulate IL-1 production. These responses are mediated by specific cell-surface receptors, the concentrations of which may also be subject to stimulated upregulation. We analyse a reduced, four-dimensional version of the model, and explore its asymptotic states. We find a variety of possible outcomes: runaway IL-1 production, multiple stable equilibria, stable limit cycles, and, exceptionally, quasi-periodic behaviour. These behaviours depend crucially on the form of the cell's response functions. The possible biological implications of these phenomena are discussed.

Arthritis, Rheumatoid↗
PubMed2000

Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones.

The chaperonins are a subgroup of oligomeric molecular chaperones; the best-studied examples are chaperonin 60 (GroEL) and chaperonin 10 (GroES), both from the bacterium Escherichia coli. At the end of the 20th century, the paradigm of chaperonins as protein folders had emerged, but it is likely that during the 21st century these proteins will come to be viewed as intercellular signals. Indeed, it is possible that the chaperonins were among the first intercellular signalling proteins to evolve. During the past few years, it has emerged that chaperonin 10 and chaperonin 60 can be found on the surface of various prokaryotic and eukaryotic cells, and can even be released from cells. Secreted chaperonins can interact with a variety of cell types, including leukocytes, vascular endothelial cells and epithelial cells, and activate key cellular activities such as the synthesis of cytokines and adhesion proteins. Much has been made of the high degree of sequence conservation among the chaperonins, particularly in terms of the immunogenicity of these proteins. However, different chaperonin 60 proteins can bind to different cell-surface receptors, including the Toll-like receptors, suggesting that this family of proteins cannot be treated as one biological entity and that several subfamilies may exist. Chaperonins have been implicated in human diseases on the basis of their immunogenicity. The finding that chaperonins can also induce tissue pathology suggests that they may play roles in infections and in idiopathic diseases such as atherosclerosis and arthritis.

Journal Article↗
PubMed2000

Inhibition of mitosis and induction of apoptosis in MG63 human osteosarcoma-derived cells in vitro by surface proteins from Actinobacillus actinomycetemcomintans.

Gentle saline extraction releases a heterogeneous mixture of proteins associated with the cell surface of Actinobacillus actinomycetemcomintans, termed the surface protein fraction (SF). Some SF components are biologically active and may modulate cell behaviour in a manner of putative importance in the aetiology of periodontitis. To further characterize this activity, the ability of the SF to induce mitosis and apoptosis in MG63 cells was investigated. Cells were plated at 10(3)-10(4) cells/cm(2) and allowed to attach before culture in the serum-free medium in the presence of 25 microg/ml SF for 2-24 h. The apoptotic and mitotic figures present were counted and the results expressed as an apoptotic or mitotic index. The apoptotic and mitotic compartments were very small, but there was an inverse correlation between mitosis and apoptosis. In control experiments the mitotic was higher than the apoptotic index, whilst in the presence of SF this was reversed. These results were confirmed using in situ end-labelling. SF, therefore, may stimulate apoptotic, but inhibit mitotic, activity in MG63 cells. This raises the possibility that components of SF might induce subtle changes in the balance between apoptosis and mitosis, which, in turn, could contribute to the progression of periodontitis.

Aggregatibacter actinomycetemcomitans↗
PubMed2000

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum.

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.

Adult↗
PubMed2000

Antibody-targeted lethal photosensitization of Porphyromonas gingivalis.

We have previously demonstrated that Porphyromonas gingivalis is susceptible to killing by toluidine blue O (TBO) when irradiated with light from a helium-neon (HeNe) laser. The aim of this study was to determine whether a TBO-antibody conjugate (Ab-TBO) could be used to specifically target P. gingivalis to lethal photosensitization in the presence of Streptococcus sanguis or human gingival fibroblasts (HGFs). When a mixture of P. gingivalis and S. sanguis was exposed to 4 microg of TBO/ml and irradiated with HeNe laser light, there were 1.5- and 4.0-log(10)-unit reductions in the viable counts, respectively. In contrast, when TBO was conjugated with a murine monoclonal antibody against P. gingivalis lipopolysaccharide, the reductions in viable counts of P. gingivalis and S. sanguis amounted to 5.0 and 0.1 log(10) units, respectively. Lethal photosensitization of P. gingivalis in the presence of HGFs using unconjugated TBO resulted in a 0.7-log(10)-unit reduction in P. gingivalis viable counts and a 99% reduction in the incorporation of tritiated thymidine ([(3)H]Tdr) by the HGFs. In contrast, when the Ab-TBO conjugate was used, there was a 100% reduction in P. gingivalis viable counts but no significant reduction in the incorporation of [(3)H]Tdr by HGFs. These results demonstrate that specific targeting of P. gingivalis can be achieved using TBO conjugated to a monoclonal antibody raised against a cell surface component of this organism.

Animals↗
PubMed2000

Identification of a novel gene cluster encoding staphylococcal exotoxin-like proteins: characterization of the prototypic gene and its protein product, SET1.

We report the discovery of a novel genetic locus within Staphylococcus aureus that encodes a cluster of at least five exotoxin-like proteins. Designated the staphylococcal exotoxin-like genes 1 to 5 (set1 to set5), these open reading frames have between 38 and 53% homology to each other. All five proteins contain consensus sequences that are found in staphylococcal and streptococcal exotoxins and toxic shock syndrome toxin 1 (TSST-1). However, the SETs have only limited overall sequence homology to the enterotoxins and TSST-1 and thus represent a novel family of exotoxin-like proteins. The prototypic gene in this cluster, set1, has been cloned and expressed. Recombinant SET1 stimulated the production of interleukin-1beta, interleukin-6, and tumor necrosis factor alpha by human peripheral blood mononuclear cells. PCR analysis revealed that set1 was distributed among other strains of S. aureus but not in the other staphylococcal species examined. Sequence analysis of the set1 genes from different strains revealed at least three allelic variants. The protein products of these allelic variants displayed a 100-fold difference in their cytokine-inducing potency. The distribution of allelic variants of the set genes among strains of S. aureus may contribute to differences in the pathogenic potential of this bacterium.

Amino Acid Sequence↗
PubMed2000

Rapid screening for putative exported proteins from Staphylococcus aureus using alkaline phosphatase as a reporter molecule.

Staphylococcus aureus causes a wide range of infections in humans, ranging from superficial skin infections to the more serious toxin-mediated diseases such as toxic shock syndrome. Owing to the increasing resistance of this bacterium to a wide range of antibiotics, the need to determine the virulence factors involved in infection is becoming more important as these molecules are potential therapeutic targets. In this study, we have screened for putative exported proteins from S. aureus on the basis that these proteins are likely to be the first point of contact between the bacterium and host during infection. We have constructed gene fusions between S. aureus DNA and a truncated version of the Escherichia coli phoA gene, and we report on the characterization of the recombinants exhibiting alkaline phosphatase activity. As well as known S. aureus proteins, we have identified a number of putative open reading frames that encode proteins similar to those from nonstaphylococcal species and also unique proteins that do not have any homologues on the current databases.

Alkaline Phosphatase↗