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This chapter focuses on the use of spinal cord stimulation (SCS) for neuronal control of heart. Myocardial ischemia evokes a myriad of responses from the heart itself, to the neurohumoral systems that modulate it, to behavioral consequences including the perception of pain. The treatment of myocardial ischemia and resultant cardiac pain has evolved from bed rest, to the advent of pharmacological and surgical approaches targeted at coronary blood flow and heart muscle, to the concept of therapies based upon modulating the interdependent interactions between the heart and its associated neurohumoral control systems. Neuromodulation therapy, using electrical stimulation of peripheral (TENS) or central (spinal cord stimulation, SCS) sites, is one such emerging therapy. It has a clinical history of over 20 years in treating cardiac pain, especially in patients refractory to conventional surgical or pharmacological approaches. Recognized patient benefits to SCS include reduced ST segment alterations induced during exercise, improved myocardial lactate metabolism, and increased workload tolerance. SCS modifies and modulates the myocytes along with the primary neural control mechanisms that regulate the heart. SCS for treatment of angina pectoris has proved to be much more effective and dependable than when used for neuropathic pain conditions. The success rate for relieving angina pectoris is often in the range of 80% or greater after several years of follow-up. Besides the reduction in angina pectoris, clinical studies have shown that SCS also reduces the ischemia associated with exertional stress while maintaining the pain response to critical levels of ischemia.

Chlamydia spp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein for Chlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectible by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of the Pseudomonas spp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extended Chlamydia T3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of the Chlamydia T3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydia neutralizing antibodies.

Interleukin-1β (IL-1β) is a proinflammatory cytokine increased in the heart following myocardial infarction. Vascular endothelial growth factors (VEGFs) are implicated in angiogenesis due to their involvement in the recruitment and proliferation of endothelial cells. Here we studied expression of VEGFs in response to IL-1β in rat cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of VEGF-D. cDNA array analysis indicated that IL-1β modulates the expression of numerous angiogenesis-related genes, notably decreasing the expression of VEGF-D, RT-PCR and Western blot analyses confirmed decreased expression of VEGF-D in response to IL-1β. IL-1β decreased the expression of VEGF-C to a lesser extent with no effects on VEGF-A or -B. Inhibition of ERK1/2, JNKs, or PKCα/β1 alone partially inhibited IL-1β-induced VEGF-D downregulation. Concurrent inhibition of ERK1/2 or JNKs and PKCα/β1 resulted in a synergistic inhibition of IL-1β-induced decreases in VEGF-D. Inhibition of ERK1/2 partially inhibited IL-1β-stimulated inactivation of GSK-3β with no effect on β-catenin levels. Inhibition of GSK-3β using SB216763 inhibited basal VEGF-D expression. We conclude that IL-1β downregulates VEGF-D expression in CMECs via the involvement of ERK1/2, JNKs, and PKCα/β1. This is the first report to indicate inhibition of VEGF-D gene expression in response to IL-1β in cardiac microvascular endothelial cells, a cell type of central interest in angiogenesis.

Genetic defects in Nucleotide excision repair (NER) lead to the clinical disorder xeroderma pigmentosum (XP) in humans which is characterized by dramatically increased sensitivity to UV light and a predisposition to development of skin cancers.1,2 NER is a major mechanism of DNA repair in cells for the removal of a large variety of bulky DNA lesions induced by environmental genotoxic agents and chemicals. The molecular basis of XP has been attributed to mutations in any of the eight XP genes, XPA through G whose products are required for NER-mediated removal of DNA damage and XP-variant (XPV). The XP proteins involved in NER can be divided into three groups based on their activity in the NER process. XPA, XPC and XPE are required for sensing DNA damage and initiating the repair process. XPB and XPD, components of the basal transcription factor TFIIH, are helicases that create a DNA strand opening surrounding the adducted base(s) during NER. XPG and XPF are the endonucleases that perform the dual incisions to release the damaged strand and allow resynthesis using the nondamaged strand as a template.3-5 Protein-protein interactions are integral for the correct assembly of the pre-incision complex and for the positioning of the nucleases prior to incision. However, these proteins have been found to form complexes with other proteins not directly involved in the NER mechanism. This chapter describes these proteins and their interactions and discusses their effects on the XP proteins, DNA repair, and genome stability.

Nectin-1, a member of the immunoglobulin superfamily, is a Ca2+-independent cell adhesion protein implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also regulates cell-cell adhesion and cell polarization in a Cdc42- and Rac-dependent manner. Western blot analyses demonstrated that accumulation of host nectin-1 is decreased by 85 % at 48 hours post-infection (h.p.i.) in Chlamydia trachomatis serovar E-infected HeLa cells. Time-course experiments demonstrated that this decrease was sustained to 60 h.p.i. Nectin-1 downregulation in C. trachomatis-infected cells was prevented by both chloramphenicol exposure and prior inactivation of the chlamydiae with UV light, demonstrating that active C. trachomatis replication was required. Penicillin G-exposure studies demonstrated that nectin-1 accumulation was also altered during persistent infection. Finally, RT-PCR analyses indicated that chlamydial infection did not alter accumulation of any nectin-1 transcripts, demonstrating that nectin-1 accumulation is reduced at a post-transcriptional level. Intesrestingly, N-cadherin-dependent cell-cell junctions can be disrupted by C. trachomatis infection, as reported by Prozialeck et al. (2002). Because interaction of nectin molecules on adjacent cells is essential for AJ formation, these data suggest that C. trachomatis may disrupt AJs, at least in part, by diminishing nectin-1 accumulation. Notably, release of chlamydiae-infected epithelial cells has been observed both in vitro from polarized monolayers and in vivo from tissues, suggesting that chlamydia-modulated downregulation of adhesion molecules and the subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis.

Several inducers of chlamydial persistence have been described, including interferon-γ (IFN-γ), IFN-α, IFN-β, and tumour necrosis factor-α (TNF-α) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-γ, IFN-α, and TNF-α are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-β, IFN-γ, indoleamine 2,3-dioxygenase, lymphotoxin-α and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C6-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.

Objectives: Premenstrual exacerbation of depression (PMED) is a variation of major depressive disorder (MDD) in which symptoms worsen during the premenstrual period. Breakthrough symptoms of PMED may occur despite effective antidepressant treatment in the rest of the cycle. This pilot study is designed to evaluate the effectiveness of variable dosing in PMED using the antidepressant sertraline. Methods: Women diagnosed with PMED were started on sertraline (up to 50 or 100 mg/day). Those subjects demonstrating continued PMED on a constant dose (n = 9) were entered into a double-blind crossover protocol, receiving either placebo or an increase in sertraline premenstrually. Each subject was evaluated twice a month (follicular and luteal phase) by clinical evaluation, Beck Depression Inventory (BDI), Hamilton Depression Rating Scale (HAM-D), and daily symptom diary and had blood collected for sertraline assay. Results: Use of variable dosing resolved the PMED, such that there was no longer a significant difference in scores for the BDI and the HAM-D between the luteal and follicular phases during treatment with sertraline supplement (p = 0.32 and p = 0.53, paired t test, respectively). However, during the months with placebo supplement, the luteal and follicular phases showed a trend for differences for the BDI (p = 0.06, paired t test) and significant differences for the HAM-D (p = 0.02 paired t test); that is, the subjects retained the PMED pattern. Sertraline levels reflected the change in dosing pattern, with higher levels seen during the luteal phase during the months with sertraline supplement but no difference seen between luteal and follicular phases during months with placebo supplement (p = 0.07 and 0.69, paired t test, respectively). Conclusions: The use of variable dosing in PMED increases the effectiveness of treatment.

The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation.