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Biomedical subjects

J Lynch

Publications and source records attributed to J Lynch.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of a muscarinic receptor antagonist via a diastereoselective Michael reaction, selective deoxyfluorination and aromatic metal-halogen exchange reaction.

An efficient synthesis of a structurally unique, novel M(3) antagonist 1 is described. Compound 1 is conveniently disconnected retrosynthetically at the amide bond to reveal the acid portion 2 and the amine fragment 3. The synthesis of key intermediate 2 is highlighted by a ZnCl(2)-MAEP complex 19 catalyzed diastereoselective Michael reaction of dioxolane 7 with 2-cyclopenten-1-one (5) to establish the contiguous quaternary-tertiary chiral centers and a subsequent geminal difluorination of ketone 17 using Deoxofluor in the presence of catalytic BF(3).OEt(2). The synthesis of the amine moiety 3 is highlighted by the discovery of a novel n-Bu(3)MgLi magnesium-halogen exchange reaction for selective functionalization of 2,6-dibromopyridine. This new and practical metalation protocol obviated cryogenic conditions and upon quenching with DMF gave 6-bromo-2-formylpyridine (26) in excellent yield. Further transformations afforded the amine fragment 3 via reductive amination with 35, Pd-catalyzed aromatic amination, and deprotection. Finally, the highly convergent synthesis of 1 was accomplished by coupling of the two fragments. This synthesis has been used to prepare multi-kilogram quantities of the bulk drug.

Amides↗
PubMed2001

Functional specialization of calreticulin domains.

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Animals↗
PubMed2001

Income inequality, the psychosocial environment, and health: comparisons of wealthy nations.

BACKGROUND: The theory that income inequality and characteristics of the psychosocial environment (indexed by such things as social capital and sense of control over life's circumstances) are key determinants of health and could account for health differences between countries has become influential in health inequalities research and for population health policy. METHODS: We examined cross-sectional associations between income inequality and low birthweight, life expectancy, self-rated health, and age-specific and cause-specific mortality among countries providing data in wave III (around 1989-92) of the Luxembourg Income Study. We also used data from the 1990-91 wave of the World Values Survey (WVS). We obtained life expectancy, mortality, and low birthweight data from the WHO Statistical Information System. FINDINGS: Among the countries studied, higher income inequality was strongly associated with greater infant mortality (r=0.69, p=0.004 for women; r=0.74, p=0.002 for men). Associations between income inequality and mortality declined with age at death, and then reversed among those aged 65 years and older. Income inequality was inconsistently associated with specific causes of death and was not associated with coronary heart disease (CHD), breast or prostate cancer, cirrhosis, or diabetes mortality. Countries that had greater trade union membership and political representation by women had better child mortality profiles. Differences between countries in levels of social capital showed generally weak and somewhat inconsistent associations with cause-specific and age-specific mortality. INTERPRETATION: Income inequality and characteristics of the psychosocial environment like trust, control, and organisational membership do not seem to be key factors in understanding health differences between these wealthy countries. The associations that do exist are largely limited to child health outcomes and cirrhosis. Explanations for between-country differences in health will require an appreciation of the complex interactions of history, culture, politics, economics, and the status of women and ethnic minorities.

Birth Weight↗
PubMed2001

Utility of a lung biopsy for the diagnosis of idiopathic pulmonary fibrosis.

It is not known if a surgical lung biopsy is necessary in all patients for the diagnosis of idiopathic pulmonary fibrosis (IPF). We conducted a blinded, prospective study at eight referring centers. Initially, cases were evaluated by clinical history and examination, transbronchial biopsy, and high-resolution lung computed tomography scans. Pulmonologists at the referring centers then assessed their certainty of the diagnosis of IPF and provided an overall diagnosis, before surgical lung biopsy. The lung biopsies were reviewed by a pathology core and 54 of 91 patients received a pathologic diagnosis of IPF. The positive predictive value of a confident (certain) clinical diagnosis of IPF by the referring centers was 80%. The positive predictive value of a confident clinical diagnosis was higher, when the cases were reviewed by a core of pulmonologists (87%) or radiologists (96%). Lung biopsy was most important for diagnosis in those patients with an uncertain diagnosis and those thought unlikely to have IPF. These studies suggest that clinical and radiologic data that result in a confident diagnosis of IPF by an experienced pulmonologist or radiologist are sufficient to obviate the need for a lung biopsy. Lung biopsy is most helpful when clinical and radiologic data result in an uncertain diagnosis or when patients are thought not to have IPF.

Biopsy↗
PubMed2001

Biomarkers for assessing occupational exposures to 1,3-butadiene.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Adult↗
PubMed2001

BD monomer and elastomer production processes.

The monomer 1,3 butadiene (BD) is a product of the petrochemical industry. It is used to make several elastomers including the very high volume styrene butadiene rubber (SBR) that comprises the bulk of automobile tires. It is also used to make polybutadiene rubber that is used in parts of tires, coatings, composites and other products. The monomer can be converted to chlorobutadiene (chloroprene) and used to make polychloroprene (neoprene). BD is one of the several olefins created by cracking hydrocarbons in the presence of steam. A mixed C4 stream from the steam cracker is then sent to a BD monomer extraction unit. Modern units typically use dimethyl formamide as the extraction solvent. SBR is commonly made by the copolymerization of BD and styrene, along with various additives to control the reaction, in a water emulsion. The reaction proceeds in a continuous chain of reactors until it is 'shortstopped' by a strong reducing agent. After removing unreacted monomers from the stabilized latex, it is blended, coagulated and dewatered. The resulting dry rubber crumb is bailed, film wrapped and stored in crates. The polymerization of BD to make polybutadiene rubber can be conducted as a water suspension type polymerization similar to SBR or in a solvent system followed by solvent recovery and transfer into water suspension.

Butadienes↗
PubMed2001

Occupational exposure to butadiene, isoprene and chloroprene.

Workers are exposed to butadiene, isoprene and chloroprene in the manufacture of these monomers and in their use in the production of various elastomers. These include styrene butadiene rubber, polybutadiene, polyisoprene, butyl rubber and neoprene. Monomer production and extraction are done in typical closed chemical process units where low background levels of the monomers are the result of minor leaks in valves and pumps. Occasionally, higher levels occur as a result of planned or unplanned events that cause releases. Polymer production is also a closed process, but the occasional clogging of pipes and equipment with polymer requiring maintenance operations where some release is likely occurs much more often than for monomer production. For this reason, exposure levels are generally higher on polymer production units. Polymer finishing is essentially an open process, but almost all monomer should have been stripped from the polymer before finishing. Where small amounts of solvents or monomers remain in the polymer and are volatilized in finishing, they are captured by vapor control systems. As a result, exposures in finishing are typically low. Measured levels of exposure in recent years are presented. In general, modern levels of exposure are well below OSHA, ACGIH and other applicable limits. Few measurements were made prior to the 1970s, but epidemiological estimates made by modeling suggest that levels could have been quite high in the 1940s and 1950s. In these years, manual reactor cleaning was common, and pumps often leaked.

Air Pollutants, Occupational↗
PubMed2001

Alternating hepatic arterial infusion and systemic chemotherapy for liver metastases from colorectal cancer: a phase II trial using intermittent percutaneous hepatic arterial access.

PURPOSE: To evaluate the objective response to a short course of hepatic arterial infusion (HAI) using temporary, percutaneously placed catheters alternating with systemic prolonged continuous infusion fluorouracil (ci 5-FU) and daily oral leucovorin (L). PATIENTS AND METHODS: Eligible patients were previously untreated (except for adjuvant therapy) adults with liver-predominant metastases, with Eastern Cooperative Oncology Group performance status of 0 to 2. Treatment regimen included HAI with fluorodeoxyuridine (FUDR) 60 mg/m2/d and L 15 mg/m2/d continuously infused daily for 4 days. After a 1-week rest, ci 5-FU was administered through a central venous access device using a dose of 180 mg/m2/d with a fixed dose of oral L at 5 mg/m2/d for 21 out of 28 days. Cycles were repeated every 6 weeks. After four cycles of therapy, patients were maintained on ci 5-FU and daily oral L until evidence of progression. RESULTS: Forty-three patients were enrolled onto this trial. One patient was ineligible. The objective response rate for all patients (17 partial, zero complete) was 41% (95% confidence interval [CI], 26% to 56%). Five patients were not able to receive at least one complete cycle of HAI. Among patients who received at least one complete cycle of HAI, the response rate was 46% (95% CI, 30% to 62%). Five patients underwent a liver resection after enrolling onto the protocol. At the time of analysis, estimated median time to progression was 6 months, and estimated median overall survival was 13 months. CONCLUSION: The objective response rate was comparable to that achieved with more prolonged and more frequent HAI using FUDR. This approach should be studied as an acceptable alternative to surgically placed hepatic arterial catheters/pumps and may have a role as neoadjuvant therapy for liver metastases that are unresectable, as well as an adjuvant role for patients with resected hepatic metastatic colorectal cancer.

Adult↗
PubMed2001

Prostate biopsy protocols: 3D visualization-based evaluation and clinical correlation.

OBJECTIVES: Systematic needle core biopsy is commonly used for the diagnosis of prostate cancer by urologists worldwide. As accurate and early diagnosis will result in more and better options for treatment, it is critical that the best possible protocols for biopsy be used clinically. In this study, we develop three-dimensional (3D) modeling and simulation technologies to evaluate most of the biopsy protocols in current clinical use, and correlate the results with those from clinical cases. MATERIALS AND METHODS: Using deformable modeling techniques, 3D computerized prostate surface models were reconstructed from step-sectioned, whole-mounted radical prostatectomy specimens with localized prostate cancer. A 3D computer simulation system was developed to accurately depict the anatomy of the prostate and all individual tumor foci. A user-friendly interface was developed in the system so that a physician can easily and interactively use it for prostate needle core biopsy. A total of 281 prostate models were reconstructed, and 18 biopsies were performed by a urologist on each model to determine the detection rates of seven different biopsy protocols. Clinical biopsies from 35 patient cases were also reviewed and correlated with the simulation results. RESULTS: The most commonly used sextant biopsy had only a 71.5% detection rate, while rates for the 10-pattern and 12-pattern protocols were much higher (96.4% and 97.2%, respectively). Even the lateral 4-pattern protocol performed better than the sextant protocol, with a detection rate of 89.3%. The lateral sextant biopsy protocol (using sites similar to, but more lateral than, those in the sextant protocol) achieved a rate of 92.5%. Although the rate of the 14-pattern biopsy was a little higher (97.5%), it used four more biopsies to achieve this increase, which, according to McNemar's test, is not statistically significant when compared to results with the 10-pattern protocol. The 5-region protocol, which uses 12 biopsies, had a detection rate of 89.7%. Transition zone and seminal vesicle biopsies did not result in a significantly increased detection rate when added to the patterns above. The clinical correlation also confirmed that the 10-pattern protocol was significantly superior to the traditional sextant biopsy pattern. CONCLUSIONS: The 10-pattern biopsy protocol was the most optimized among all the protocols evaluated. This protocol supplemented the sextant biopsy protocol with four more lateral biopsies in the mid and apical sites on both sides.

Biopsy, Needle↗
PubMed2001

The health economic implications of treatment with quetiapine: an audit of long-term treatment for patients with chronic schizophrenia.

This retrospective, case series audit assessed the clinical and health-economic impact of long-term treatment with quetiapine ('Seroquel'), a new atypical antipsychotic, in patients with chronic schizophrenia. The study design was of a case series format, comprising patients entered from one centre into the open-label extension of a multicentre 6-week efficacy study. Twenty-one patients (15 male, six female; mean age 39 years) were studied, of whom 17 (81%) had been rated as 'partially responsive' to previous antipsychotics. Data on hospitalisations and information on symptoms were collected retrospectively for the 12 months before quetiapine treatment was initiated and for the 12 months after. Quetiapine was effective in reducing psychotic symptoms with mean BPRS scores reducing significantly, from 38 to 21 (P < 0.005). Motor function was also significantly improved with mean Simpson scale scores reducing from 15 to 12 (P < 0.005). Average inpatient days were reduced by 11% in year two (97 compared with 109 days) while the overall costs of treatment, including drug costs, fell by 5% (I pound sterling 20,843 to I pound sterling 19,827). Four patients had been hospitalised for longer than 5 years before starting quetiapine; these chronically institutionalised patients remained in hospital, despite improved clinical outcomes (mean BPRS scores after treatment of 34, compared with 43 before), for the full 12 months of quetiapine treatment. Were the data from this audit to be re-analysed excluding these four patients then average inpatient days would have been reduced by 33% (45 to 30 days) and overall cost of treatment by 19% (I pound sterling 8617 to I pound sterling 7011). This audit suggests that treatment with quetiapine over this 1-year period was associated with both clinical improvements and a decreased usage of inpatient services. The reduction in hospitalisation costs would appear to compensate for the increased cost of drug treatment. Significantly, potential savings appear to be greatest for those patients with a 'revolving door' pattern of repeated readmission.

Adult↗
PubMed2001

Recombinant human thrombopoietin (rhTPO) after autologous bone marrow transplantation: a phase I pharmacokinetic and pharmacodynamic study.

Thrombocytopenia following myelotoxic therapy is a common problem and when severe (<20,000/microl) can lead to severe morbidity and mortality. Thrombopoietin (TPO) is a naturally occurring glycosylated peptide which stimulates the differentiation of bone marrow stem cells into megakaryocyte progenitor cells, induces the expression of megakaryocyte differentiation markers, promotes megakaryocyte proliferation, polyploidization and, ultimately, the formation of increased numbers of platelets in the circulation. TPO has now been produced by recombinant technology and has entered clinical trials. This open label phase I study was designed to determine the safety, tolerance and pharmacokinetics of recombinant thrombopoietin (rhTPO) when administered to patients after undergoing high-dose chemotherapy followed by autologous bone marrow transplantation. rhTPO was administered intravenously by bolus injection at doses ranging from 0.3 to 4.8 microg/kg/day every 3 days to 30 patients and 0.6 microg/kg daily to three patients. rhTPO was begun the day after marrow infusion and continued until platelet recovery to >20,000/microl. G-CSF was concomitantly administered to promote myeloid recovery. Serious adverse events or neutralizing antibodies to rhTPO were not observed during the study. Median platelet recovery after ABMT was 19 days (range, 11-41). Neither the dose nor the schedule of rhTPO appeared to have any impact upon the time course of platelet recovery. In this phase I study, rhTPO was found to be well tolerated without the development of neutralizing antibodies and without compromising neutrophil recovery. Platelet recovery was similar for all doses studied warranting further evaluation in phase II and III trials designed to test for platelet recovery efficacy.

Adult↗
PubMed2001

Progenitor and lymphoma cells in blood stem cell harvests: impact on survival following transplantation.

This study evaluated whether cytokine-induced blood stem cell mobilization also mobilized lymphoma cells and whether lymphoma cell mobilization affected outcome post autologous blood stem cell transplant. Blood stem cell collections from 26 non-Hodgkin's lymphoma (NHL) patients harvested during steady-state (non-mobilized) and from 35 NHL patients harvested after cytokine administration (mobilized) were studied. The harvests were cultured and molecularly evaluated for clonal markers of the primary lymphoma. All patients underwent high-dose chemotherapy and autologous transplantation. Graft products from mobilized patients were more likely to contain lymphoma than graft products from non-mobilized patients (37% vs 19%) but this difference was not significant (P = 0.16). In a multivariate analysis, lymphoma contamination was not associated with patient age, gender, tumor grade, prior radiotherapy, duration of prior chemotherapy, mononuclear cell count, or the number of aphereses performed to obtain the product. Heavily pre-treated patients were less likely to have lymphoma-contaminated harvests (P = 0.064). Lymphoma contamination was positively associated with the number of progenitor cells collected (P = 0.047). In multivariate analyses, the only significant independent predictor of lymphoma contamination was the number of mononuclear cells collected (P = 0.031). Lymphoma contamination of transplanted apheresis products had no apparent impact on event-free and overall survival.

Adult↗
PubMed2001

Extracellular superoxide dismutase overexpression improves behavioral outcome from closed head injury in the mouse.

Oxidative stress is known to play an important role in the response of brain to traumatic insults. We tested the hypothesis that increased extracellular superoxide dismutase (EC-SOD) expression can reduce injury in a mouse model of closed head injury. Neurologic, cognitive, and histologic outcomes were compared between transgenic mice exhibiting a fivefold increase in EC-SOD activity and wild-type littermate controls. Severe or moderate transcranial impact was induced in anesthetized and physiologically controlled animals. After severe impact, transgenic mice had better neurological outcome at 24 hr postinjury (p = 0.038). Brain water content was increased, but there was no difference between groups. Moderate impact resulted in predominantly mild neurologic deficits in both groups at both 24 hr and 14 days postinjury. Morris water maze performance, testing cognitive function at 14-17 days after trauma, was better in EC-SOD overexpressors (p = 0.018). No differences were observed between groups for histologic damage in hippocampal CA1 and CA3. We conclude that EC-SOD has a beneficial effect on behavioral outcome after both severe and moderate closed head injury in mice. Because EC-SOD is believed to be predominantly located in the extracellular space, these data implicate an adverse effect of extracellular superoxide anion on outcome from closed head injury.

Animals↗
PubMed2001

Women's exposure to early and later life socioeconomic disadvantage and coronary heart disease risk: the Stockholm Female Coronary Risk Study.

BACKGROUND: Measures of low socioeconomic position have been associated with increased risk for coronary heart disease (CHD) among women. A more complete understanding of this association is gained when socioeconomic position is conceptualized from a life course perspective where socioeconomic position is measured both in early and later life. We examined various life course socioeconomic indicators in relation to CHD risk among women. METHODS: The Stockholm Female Coronary Risk Study is a population-based case-control study, in which 292 women with CHD aged < or =65 years and 292 age-matched controls were investigated using a wide range of socioeconomic, behavioural, psychosocial and physiological risk factors. Socioeconomic disadvantage in early life (large family size in childhood, being born last, low education), and in later life (housewife or blue-collar occupation at labour force entry, blue-collar occupation at examination, economic hardships prior to examination) was assessed. RESULTS: Exposure to early (OR = 2.65, 95% CI : 1.12-6.54) or later (OR = 5.38, 95% CI : 2.01-11.43) life socioeconomic disadvantage was associated with increased CHD risk as compared to not being exposed. After simultaneous adjustment for marital status and traditional CHD risk factors, early and later socioeconomic disadvantage, exposure to three instances of socioeconomic disadvantage in early life was associated with an increased CHD risk of 2.48 (95% CI : 0.90-6.83) as compared to not being exposed to any disadvantage. The corresponding adjusted risk associated with exposure to later life disadvantage was 3.22 (95% CI : 1.02-10.53). Further analyses did not show statistical evidence of interaction effects between early and later life exposures (P = 0.12), although being exposed to both resulted in a 4.2-fold (95% CI : 1.4-12.1) increased CHD risk. Exposure to cumulative socioeconomic disadvantage (combining both early and later life), across all stages in the life course showed strong, graded associations with CHD risk after adjusting for traditional CHD risk factors. Stratification of cumulative disadvantage by body height showed that exposure to more than three periods of cumulative socioeconomic disadvantage had a 1.7- (95% CI : 0.9-3.2) and 1.9- (95% CI : 1.0-7.7) fold increased CHD risk for taller and shorter women, respectively. The combination of both short stature and more than two periods of cumulative socioeconomic disadvantage resulted in a 4.4-fold (95% CI : 1.7-9.3) increased CHD risk. CONCLUSIONS: Both early and later exposure to socioeconomic disadvantage were associated with increased CHD risk in women. Later life exposure seems to be more harmful for women's cardiovascular health than early life exposure to socioeconomic disadvantage. However, being exposed to socioeconomic disadvantage in both early and later life magnified the risk for CHD in women. Cumulative exposure to socioeconomic disadvantage resulted in greater likelihood of CHD risk, even among women who were above median height. In terms of better understanding health inequalities among women, measures of socioeconomic disadvantage over the life course are both conceptually and empirically superior to using socioeconomic indicators from one point in time.

Adult↗
PubMed2001

Cerebral blood flow velocity response to magnesium sulfate in patients after subarachnoid hemorrhage.

Magnesium sulfate therapy, standard in preventing seizures in preeclampsia, is under active investigation as a neuroprotective agent. The authors studied the effect of magnesium as a cerebral vasodilator by measuring the cerebral blood flow velocity (CBFV) response to a 5g intravenous bolus of MgSO4 compared with a saline placebo after subarachnoid hemorrhage (SAH). Transcranial Doppler ultrasonography of the middle cerebral artery (MCA) was measured after each infusion. Patients were studied up to three times after SAH at prescribed time intervals. Fourteen patients (11 women, 3 men; mean age 58 years) underwent 29 studies. All patients underwent hypertensive, hypervolemic therapy. Four patients developed cerebral vasospasm. Doubling serum magnesium levels did not affect MCA CBFV but slightly lowered mean arterial blood pressure and systemic vascular resistance. Intravenous magnesium bolus did not reduce elevated CBFV in the subset of SAH patients with clinical vasospasm. The role of magnesium sulfate as a cerebral vasodilator in patients with SAH requires further study.

Adult↗
PubMed2001

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

Animals↗
PubMed2001

Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63.

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.

Aspartic Acid↗