Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility.

BACKGROUND: A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome. OBJECTIVES: A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease. STUDY DESIGN: A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated. RESULTS: The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus. CONCLUSIONS: The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.     

Correlation of penicillin Binding protein 2a detection with oxacillin resistance in Staphylococcus aureus and discovery of a novel penicillin binding protein 2a mutation

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in Staphylococcus aureus. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of S. aureus. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The mecA gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 microg/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 microg/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the mecA gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4+) or repeatedly weak (1 to 2+) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant S. aureus and facilitated the discovery of a novel, functionally impaired form of PBP2a.     

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents

Kv4.3 channels conduct transient outward K⁺ currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K⁺ channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K⁺ currents.     

Mice with mutations in Mahogunin ring finger-1 (Mgrn1) exhibit abnormal patterning of the left-right axis.

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20-25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46-60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis.     

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma

Chromosomal rearrangements of the MYC locus, which often involve the IG loci, are recurrent events in multiple myeloma (MM) and plasma cell leukemia (PCL). We used dual-color fluorescence in situ hybridization (FISH) to characterize the breakpoint locations of chromosomal translocations/rearrangements involving the MYC locus at 8q24 found in a panel of 14 MM cell lines and 70 primary tumors (66 MM and 4 PCL). MYC locus alterations were observed in 21 cases: MYC/IG (mainly IGH@) fusions in 11 cell lines and three patients (2 MM and 1 PCL), and extra signals and/or abnormal MYC localizations in seven patients (5 MM and 2 PCL). Fourteen of these cases were investigated by FISH analyses by use of a panel of BAC clones covering about 6 Mb encompassing the MYC locus. The breakpoints were localized in a region 100-250 kb centromeric to MYC in four cases, a region 500-800 kb telomeric to the gene in four cases, and regions > or = 2 Mb centromeric or telomeric to MYC in five cases. Two different breakpoints were detected in the KMS-18 cell line, whereas the insertion of a MYC allele was found in a complex t(16;22) chromosomal translocation in the RPMI8226 cell line. Our data document a relatively high dispersion of 8q24 breakpoints in MM.     

The N-terminal domain of the vaccinia virus E3L-protein is required for neurovirulence, but not induction of a protective immune response

Encephalitis is a rare, but serious complication from vaccination against smallpox using replication competent strains of vaccinia virus. In this report we describe mutants of vaccinia virus, containing N-terminal deletions of the vaccinia virus interferon resistance gene, E3L, that are attenuated for neuropathogenesis in a mouse model system. These recombinant viruses replicated to high titers in the nasal mucosa after intra-nasal infection of C57BL/6 mice but failed to spread to the lungs or brain. These viruses demonstrated reduced pathogenicity after intra-cranial infection as well, indicating a decrease in neurovirulence. Intra-nasal inoculation or inoculation by scarification with a low dose of recombinant virus containing a deletion of the entire N-terminal domain of E3L protected against challenge with a high dose of wild-type vaccinia virus, suggesting that this replication competent, but attenuated strain of vaccinia virus may have promise as an improved vaccine for protecting against smallpox, and as a vector for inducing mucosal immunity to heterologous pathogenic organisms.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

Investigation of Gamma-aminobutyric acid (GABA) A receptors genes and migraine susceptibility

Background  

Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24–28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ), which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24–28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type ε (GABRE) and type θ (GABRQ) genes and their involvement in migraine.     

Complex Acetylenes from the Roots of Ferula communis

The benzene extract from the roots of FERULA COMMUNIS afforded the coumarin ferulenol ( 1) and the structurally related acetylenes named ferulinolone [3-(1,2-dihydrofalcarinolonyl)-ferulenol] ( 4) and decarboxyferulinolone [2-nor-3-(1,2-dihydrofal-carinolonyl-ferulenol] ( 7). The structures of these substances were deduced from their spectral data and those of their derivatives, mainly from the (1)H- and (13)C-NMR 1D and 2D spectra (one bond and long-range correlations). Methoxylatifolone ( 10) was also isolated from this extract.