A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.Nucleic acid detection techniques such as PCR provide the potential for rapid and sensitive detection of serious, treatable virus infections, such as those caused by the herpes group of viruses. Detection of members of this group may comprise up to half the workload of many diagnostic virology laboratories. Virus isolation, shell vial-based assays, cytomegalovirus (CMV) pp65 antigenemia assays, and immunofluorescence (IF) assays all suffer from one or more limitations, including, respectively, slowness, insensitivity when applied to blood specimens, lack of suitability for high specimen throughput, and a requirement for infected cells in the specimen. PCR has the potential to overcome each of these limitations and also has applicability over a wide range of specimen types. Multiplex PCR assays have the additional advantage of combining primers that are specific for viruses associated with several potential differential diagnoses in the one test, thereby offering increased efficiency and cost-effectiveness. Multiplex PCR assays have been described for herpesviruses, although they vary in terms of the virus types represented and the specimens analyzed. For example, assays for the simultaneous detection of varicella-zoster virus (VZV), herpes simplex viruses (HSV), CMV, human herpesvirus 6, and Epstein-Barr virus in cerebrospinal fluid (CSF) (11) and assays for HSV and VZV in mucocutaneous specimens (6, 9) and CSF (12) have been reported, each with improved utility over existing methods in the diagnostic setting.Our laboratory serves as a reference virus identification laboratory for a population of nearly 4 million people. A wide range of specimen types are received on a daily basis from hospital in-and outpatients as well as from those being served by general practitioners and doctors in specialized infectiousdisease clinics. These patients have diverse clinical symptoms, including ...

The 'cross-talk' between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMPdependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT-PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: a, bI, bII, c; novel: d, e, g, h) and protein kinase D (PKCl) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca 2+ -independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö 6976, and Gö 6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.

The antianginal agent perhexiline inhibits rat cardiac carnitine palmitoyltransferase-1 (CPT-1) and CPT-2, key enzymes for mitochondrial transport of long-chain fatty acids. We tested the hypothesis that perhexiline, in therapeutic concentrations (2 microM), inhibits palmitate oxidation and enhances glucose oxidation in isolated rat cardiomyocytes and in the working rat heart, thereby increasing efficiency of oxygen utilization. In isolated cardiomyocytes, perhexiline (2 microM) exerted no acute effects on palmitate oxidation, but after 48 hours pre-exposure oxidation was inhibited by perhexiline (2 to 10 microM) by 15% to 35% (P < 0.0002). In non-ischemic working rat hearts (3%BSA, 0.4 mM palmitate, 11 mM glucose, 100 microU/mL insulin) perhexiline (2 microM) had no significant acute effect on cardiac efficiency, palmitate or glucose oxidation, but 24 hours pretreatment with transdermal perhexiline increased cardiac work (by 29%, P < 0.05) and cardiac efficiency (by 30%, P < 0.02) without significant effects on palmitate oxidation. The selective CPT-1 inhibitor oxfenicine (2 mM) inhibited palmitate oxidation and enhanced glucose oxidation, but failed to enhance cardiac efficiency. In conclusion, in the non-ischemic working rat heart, perhexiline increases myocardial efficiency by a mechanism(s) that is largely or entirely independent of its effects on CPT. Effects on cardiac efficiency during ischemia, and with changes in fatty acid oxidation after longer perhexiline pretreatment remain to be determined.