Serum uric acid is not independently associated with plasma renin activity and plasma aldosterone in hypertensive adults.

BACKGROUND AND AIMS: In experimental investigations conducted in rats, raising serum uric acid (SUA) levels resulted in the stimulation of intrarenal renin expression. Studies in humans exploring the association of SUA with plasma renin activity (PRA) yielded conflicting results. Moreover, little is known about the relationship of SUA with plasma aldosterone concentration (PAC). The study aimed to assess the relationship between SUA levels, PRA, and PAC and the influence of age, gender, body mass index (BMI), and hyperuricemia on these relationships in subjects with essential hypertension (EH). METHODS AND RESULTS: We enrolled 372 hypertensive patients (mean age 45 ± 12 years, men 67%) with uncomplicated EH that was not pharmacologically treated. The study population was divided in tertiles according to SUA levels. While PRA did not differ significantly across the three tertiles, PAC was higher in subjects belonging to the uppermost tertile of SUA than those in the lower ones (p = 0.0429); however, this difference lost statistical significance after adjustment for age, sex, BMI, and serum creatinine. Univariate correlation analyses showed significant associations of SUA with PRA (r = 0.137; p = 0.008) and PAC (r = 0.179; p < 0.001). However, these relationships were not significant after correcting for confounding factors in multiple linear regression analyses. We did not observe statistically significant effect modification by gender, age, BMI, and hyperuricemia. CONCLUSION: SUA levels are weakly associated with PRA and PAC in adults with untreated EH. These relationships were lost after adjustment for age, sex, BMI, and serum creatinine.

Vitamin D receptor gene polymorphisms and plasma renin activity in essential hypertensive individuals.

Several studies analyzed 25-hydroxyvitamin D (25[OH]D) and blood pressure (BP) relationship with mixed results. Moreover, a relationship between the risk of hypertension and vitamin D receptor (VDR) gene polymorphisms, FokI and BsmI, was reported. This study was aimed to analyze these relationships in essential hypertensive (EH) patients. Seventy-one EH patients, 18-75 years old, were enrolled. Patients underwent clinical BP, 24-h ambulatory BP monitoring, 25[OH]D and plasma renin activity (PRA) evaluations. FokI and BsmI VDR polymorphisms were analyzed and compared with those of 72 healthy controls. In EH patients, the median 25[OH]D levels were lower than 30 ng ml(-1). We found a significant negative correlation between 25[OH]D and 24-h systolic BP (r = -0.277, P = 0.043). This correlation persisted in backward stepwise multivariate analyses (ß = -0.337; P = 0.022), after adjustment for age, gender, body mass index, glomerular filtration rate, and PRA. We did not observe statistically significant correlation between 25[OH]D and PRA. We compared the allelic frequencies and genotype distribution between patients and controls, and FokI and BsmI VDR polymorphisms were not associated either with hypertensive status or with PRA. Further wide studies are needed to clarify this relationship.

Relationships between mild hyperuricaemia and aortic stiffness in untreated hypertensive patients.

BACKGROUND AND AIMS: Clinical studies exploring the relationship between serum uric acid (SUA) and arterial stiffness yielded conflicting results. Only in a few of these studies, arterial distensibility was examined by measuring aortic pulse wave velocity (PWV), which is considered the gold standard for evaluating arterial stiffness. In none of the previous investigations was the influence of SUA on aortic distensibility assessed, taking into account the effect of albuminuria. The purpose of our study was to comprehensively analyse the relationships between SUA and aortic PWV in a group of essential hypertensive patients. METHODS AND RESULTS: We enrolled 222 untreated and uncomplicated hypertensive subjects (mean age: 44 ± 10 years; 60% males), without gout. In all patients, SUA and urinary albumin excretion rate (AER) were determined. Moreover, carotid-femoral (c-f) PWV was measured. C-f PWV was significantly higher in hypertensive patients belonging to the uppermost tertile of SUA distribution, compared to subjects of the lowest tertiles (10.9 ± 2.2 vs. 10 ± 1.8 vs. 9.9 ± 1.7 m s(-1); p = 0.001). In univariate analysis, SUA correlated with c-f PWV (r = 0.24; p < 0.001). This association disappeared when AER was added in a multiple regression model, including SUA, age, mean arterial pressure, gender, metabolic syndrome components and glomerular filtration rate. CONCLUSION: The results of our study showed that, in essential hypertensive subjects, there is a positive relationship between mild hyperuricaemia and aortic stiffness. This association weakened after adjustment for covariates and lost statistical significance after further correction for albuminuria.

Absence of an independent association between serum uric acid and left ventricular mass in Caucasian hypertensive women and men.

BACKGROUND AND AIM: Experimentally uric acid may induce cardiomyocyte growth and interstitial fibrosis of the heart. However, clinical studies exploring the relationship between serum uric acid (SUA) and left ventricular (LV) mass yielded conflicting results. The aim of our study was to evaluate the relationships between SUA and LV mass in a large group of Caucasian essential hypertensive subjects. METHODS AND RESULTS: We enrolled 534 hypertensive patients free of cardiovascular complications and without severe renal insufficiency. In all subjects routine blood chemistry, including SUA determination, echocardiographic examination and 24 h ambulatory blood pressure (BP) monitoring were obtained. In the overall population we observed no significant correlation of SUA with LV mass indexed for height(2.7) (LVMH(2.7)) (r = 0.074). When the same relationship was analysed separately in men and women, we found a statistically significant correlation in female gender (r = 0.27; p < 0.001), but not in males (r = -0.042; p = NS). When we grouped the study population in sex-specific tertiles of SUA, an increase in LVMH(2.7) was observed in the highest tertiles in women (44.5 ± 15.6 vs 47.5 ± 16 vs 55.9 ± 22.2 g/m(2.7); p < 0.001), but not in men. The association between SUA and LVMH(2.7) in women lost statistical significance in multiple regression analyses, after adjustment for age, 24 h systolic BP, body mass index, serum creatinine and other potential confounders. CONCLUSIONS: Our findings do not support an independent association between SUA and LV mass in Caucasian men and women with arterial hypertension.

Construction, expression, and characterization of a baculovirally expressed catalytic domain of human matrix metalloproteinase-9.

We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.

DNA and ATP binding activities of the baculovirus DNA helicase P143.

P143 is a DNA helicase that tightly binds both double-stranded and single-stranded DNA. DNA-protein complexes rapidly dissociated in the presence of ATP and Mg(2+). This finding suggests that ATP hydrolysis causes a conformational change in P143 which decreases affinity for DNA. This supports the model of an inchworm mechanism of DNA unwinding.

3'-end formation of baculovirus late RNAs.

Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mechanism of 3'-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3'-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3' ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism.

The Autographa californica nuclear polyhedrosis virus p143 gene encodes a DNA helicase.

The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a K(m) of 60 microM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.

Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement.

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.

Guanylyltransferase activity of the LEF-4 subunit of baculovirus RNA polymerase.

The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that transcribes viral late genes. This polymerase is composed of four equimolar subunits, LEF-4, LEF-8, LEF-9, and p47. Here we present data indicating that the LEF-4 subunit of RNA polymerase is a guanylyltransferase. Incubation of RNA polymerase in the presence of divalent cation and radiolabeled GTP resulted in the formation of a covalent enzyme-guanylate complex that comigrated with the LEF-4 subunit. The label transfer assay showed an absolute requirement for divalent cation which could be satisfied by either manganese or magnesium. The reaction was specific for guanine nucleotides, and GTP was more effective than dGTP in the formation of enzyme-guanylate complex. To demonstrate that LEF-4 was the guanylyltransferase, the single subunit was overexpressed in baculovirus-infected cells. The overexpressed protein was primarily cytosolic, indicating that other proteins in the RNA polymerase complex were responsible for nuclear targeting of LEF-4. LEF-4 alone was able to covalently bind GMP, although less efficiently than viral RNA polymerase.

The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity.

A virus-encoded RNA polymerase purified from baculovirus-infected cells.

A DNA-dependent RNA polymerase was purified to homogeneity, starting from insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The purified polymerase supported accurate and specific transcription from late and very late promoters but was not active on viral early promoters. Thus, promoter recognition is an integral function of the purified enzyme. The purified RNA polymerase was composed of only four equimolar subunits, which makes it the simplest DNA-directed RNA polymerase from a eukaryotic source described so far. Amino-terminal protein sequencing, peptide fingerprinting, and immunochemical analyses were used to identify the four subunits, all of which are virus encoded. Overexpression of the four viral proteins (LEF-8, LEF-4, LEF-9, and p47) in baculovirus-infected cells resulted in a significant increase in the levels of RNA polymerase produced in the infected cells. Thus, the overexpression data are consistent with our identification of the RNA polymerase subunits.

Mapping of ORF121, a factor that activates baculovirus early gene expression.

The protein product of the 39k gene of Autographa californica nuclear polyhedrosis virus is thought to be important for viral replication because of its association with the virogenic stroma and its role in activation of late gene expression Transient expression assays showed that addition of a DNA fragment encoding a 58-amino-acid polypeptide increased expression of a 39k reporter plasmid. This stimulation was dependent on cotransfection of a plasmid encoding IE1. Cotransfection of this gene, orf121, also stimulated ie1 expression, and the activation of ie1 was even more dramatic in the presence of IE1. These data suggested that ORF-121 stimulated 39k expression by upregulation of IE1 expression. Activation of 39k by ORF121 and the viral transcription factor IE2 was additive, while activation by ORF-121 and the apoptotic suppressor P35 was synergistic. Cotransfection of p39cat and pIE1 with plasmids encoding ORF121, IE2, and P35 stimulated 39cat expression more than 100-fold compared to cells transfected with only p39cat and pIE1. These data suggest that IE2 and ORF121 work by similar mechanisms and indirectly activate p39cat by increasing IE1 expression, while P35 increases 39cat expression by a different mechanism.

Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells.

The baculovirus protein IE1 is required for the transactivation of many early viral genes in transient expression assays. However, cycloheximide inhibition studies have failed to reveal a dependence of early gene transcription on expression of IE1 in infected cells. We show here that synthesis of IE1 was not effectively inhibited by the addition of 100 microg/ml cycloheximide, the concentration routinely used in these studies. However, when cycloheximide was added at 250 microg/ml, IE1 synthesis was repressed to less than 5% of control levels. These more stringent conditions were used to discriminate between immediate early and delayed early genes. Transcription of three immediate early genes (ie1, ie2, and ie0) was increased by the addition of high concentrations of cycloheximide. However, transcription of three other early genes (39k, p35, and lef-3), which are known to be dependent on IE1 transactivation, was significantly reduced by the addition of 250 microg/ml cycloheximide. Immunoblot analyses also revealed a difference between the immediate and delayed early class of viral genes. Synthesis of IE1, IE2, and IE0 was resistant to cycloheximide treatment, while translation of SSB/LEF-3 and pp31 was strongly inhibited even at the lower concentration of cycloheximide. Although cycloheximide was shown to be useful in defining early temporal classes, it induced apoptosis in both uninfected and infected Sf9 cells when used at the inhibitory concentration.

Dynamic phosphorylation of Autographa californica nuclear polyhedrosis virus pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.

Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirus ie1 promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36-48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate-early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirus ie1 promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Mapping functional domains in AcMNPV pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.

The viral ubiquitin gene of Autographa californica nuclear polyhedrosis virus is not essential for viral replication.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a protein with significant homology to ubiquitin. To study the role of viral ubiquitin in infection, a recombinant virus was constructed with a frameshift mutation within the coding sequence of the viral ubiquitin gene, v-ubi. This recombinant, named Vubi-FS, was viable, indicating that viral ubiquitin is not essential for replication in tissue culture. However, the yields of infectious budded virus were decreased 5- to 10-fold in single step growth curves, and the production of total budded virions was reduced to a similar extent. The mutant virus particles contained the phospholipid-modified form of ubiquitin (Pt-Ub), and amino acid sequence analysis revealed that only host ubiquitin was packaged into virions. Together, these results suggest that viral ubiquitin is a nonessential protein that may confer a slight growth advantage under certain conditions.

Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica.

The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.