Biomedical subjects

Jerome P Trzeciakowski

Publications and source records attributed to Jerome P Trzeciakowski.

7 recordsLinked to original sources

PubMed2006

Borrelia burgdorferi alters its gene expression and antigenic profile in response to CO2 levels.

The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO(2), serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO(2), 3% H(2), 0.087 ppm O(2)) and microaerophilic (1% CO(2), 3.48 ppm O(2)) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO(2). In this study we identified CO(2) as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO(2), independent of O(2) levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO(2). Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO(2), indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO(2) may be an additional cue for borrelial host adaptation and gene regulation.

Anaerobiosis↗

PubMed2006

GABAergic miniature postsynaptic currents in septal neurons show differential allosteric sensitivity after binge-like ethanol exposure.

Binge-like ethanol treatment of septal neurons blunts GABAAR-mediated miniature postsynaptic currents (mPSCs), suggesting it arrests synaptic development. Ethanol may disrupt postsynaptic maturation by blunting feedback signaling through immature GABAARs. Here, the impact of ethanol on the sensitivity of mPSCs to zolpidem, zinc and 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha-OH-DHP) was tested. The decay phase of mPSCs showed concentration-dependent potentiation by zolpidem (0.03-100 microM), which was substantially blunted after ethanol exposure. Since zolpidem potentiation exhibited a substantial age-dependent increase in untreated neurons, this finding supported the idea that ethanol arrests synaptic development. GABAAR alpha1 subunit protein also increased with age in untreated neurons, paralleling enhanced sensitivity to zolpidem. Surprisingly, alpha1 levels were not reduced by binge ethanol even though mPSCs were relatively zolpidem-insensitive. Zinc (3-30 microM) decreased mPSC parameters in a concentration- and age-related manner with older untreated cells showing less inhibition. However, there was no increase in mPSC zinc sensitivity after binge ethanol as would be expected if a general arrest of synaptic maturation had occurred. 3alpha-OH-DHP (3-1000 nM) induced concentration-dependent potentiation of mPSC decay. Although potentiation was age-independent, binge ethanol treatment exaggerated sensitivity to this neurosteroid. Finally, chronic picrotoxin pretreatment (100 microM) intended to mimic GABAAR inhibition from ethanol pretreatment did not significantly change mPSC modulation by zolpidem, zinc or 3alpha-OH-DHP. These results suggest that binge ethanol treatment selectively arrests a subset of processes important for maturation of postsynaptic GABAA Rs. However, it is unlikely that ethanol causes a broad arrest of postsynaptic development through a direct inhibition of GABAAR signaling.

Age Factors↗

PubMed2006

Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi.

Borrelia burgdorferi, the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surface-exposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi. Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

Adhesins, Bacterial↗

PubMed2005

Mechanical properties of the interaction between fibronectin and alpha5beta1-integrin on vascular smooth muscle cells studied using atomic force microscopy.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Animals↗

PubMed2005

Histamine effects on endothelial cell fibronectin interaction studied by atomic force microscopy.

Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.

Animals↗

PubMed2004

Binge ethanol exposure delays development of GABAergic miniature postsynaptic currents in septal neurons.

Whole cell GABA(A)R currents of septal neurons isolated from rat pups increase rapidly during the first weeks of life when inhibitory synapses are forming. Early postnatal binge ethanol intubation on days 4-9 delays this maturational up-regulation in septal neurons isolated several days later suggesting inhibitory synapse formation could be disrupted [S.-H. Hsiao, J.L. Acevedo, D.W. DuBois, K.R. Smith, J.R. West, G.D. Frye, Early postnatal ethanol intubation blunts GABA(A) receptor upregulation and modifies 3alpha-hydroxy-5alpha-pregnan-20-one sensitivity in rat MS/DB neurons, Brain Res. Dev. Brain Res. 130 (2001) 25-40]. Surprisingly, whole cell GABA(A)R function does not increase rapidly when septal neurons are grown for the same period in vitro and is not blunted by comparable ethanol exposure of the cultures [S.-H. Hsiao, D.W. DuBois, R.C. Miranda, G.D. Frye, Critically timed ethanol exposure reduces GABA(A)R function on septal neurons developing in vivo but not in vitro, Brain Res Dev. Brain Res. 1008 (2004) 69-80]. Because GABAergic miniature postsynaptic currents (mPSCs) show parallel patterns of maturation whether cortical neurons are growing in vivo or in vitro [D.D. Dunning, C.L. Hoover, I. Soltesz, M.A. Smith, D.K. ODowd, GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons, J. Neurophysiol. 82 (1999) 3286-3297], we examined the impact of binge ethanol exposure on synaptic receptors activated by these currents in septal cultures. Binge ethanol treatment of embryonic septal neurons over 6-11 days in vitro (DIV) slightly reduced GABA(A)R-mediated mPSC amplitude and frequency, but also substantially slowed decay kinetics when mPSCs were recorded later on DIV 13-18. Decreased frequency and slowed mPSC decay kinetics after ethanol were consistent with parameters measured in immature neurons. Untreated septal neurons exhibited decreased mPSC amplitude and frequency with acute 30-100 mM ethanol, without changing decay kinetics suggesting a direct inhibition of postsynaptic receptors. Sustained inhibition of GABA(A)Rs with 100 microM picrotoxin on DIV 6-11 decreased mPSC amplitude and frequency and slowed decay kinetics similar to binge ethanol exposure. These results suggest that binge ethanol exposure delays mPSC maturation by interfering with trophic postsynaptic GABA(A)R signaling during the early development of septal neurons.

Alcohol-Induced Disorders, Nervous System↗

PubMed2002

alpha(4)beta(1) Integrin activation of L-type calcium channels in vascular smooth muscle causes arteriole vasoconstriction.

A pathway for the regulation of vascular tone appears to involve coupling between integrins and extracellular matrix proteins or their fragments and the subsequent modulation of ion movement across the smooth muscle cell membrane. Here, we report that the activation of L-type voltage-activated Ca(2+) channels occurs through a novel interaction of alpha(4)beta(1) integrin with peptides containing the Leu-Asp-Val (LDV) integrin--binding sequence, which is found in the CS-1 region of an alternately spliced fibronectin variant. Experiments were conducted on arterioles isolated from rat skeletal muscle. Arterioles exhibited sustained concentration-dependent vasoconstriction to LDV peptides but not to Leu-Glu-Val (LEV) control peptides. The constriction was associated with increased smooth muscle cell [Ca(2+)](i), as measured by using fura 2. The response could be inhibited with a function-blocking anti--alpha(4) integrin antibody. Removal of the endothelium did not alter the vasoconstrictor response. Further experiments demonstrated that the vasoconstriction was abolished by the L-type Ca(2+) channel inhibitor nifedipine and the Src family kinase inhibitor PP2. In studies of isolated smooth muscle cells using whole-cell patch-clamp methods, the L-type current was enhanced by the LDV but not LEV peptide and was blocked by PP2 or antibodies to alpha(4) integrin. Collectively, these data indicate that activation of alpha(4)beta(1) integrin leads to enhanced influx of Ca(2+) through L-type channels by activating a tyrosine kinase pathway, leading to vasoconstriction. Involvement of integrins in the modulation of vascular tone may be particularly important in vascular responses to mechanical signals, such as pressure and flow, and to tissue injury after damage to the extracellular matrix.

Alternative Splicing↗