Clinical response to ertapenem in severe community-acquired pneumonia: a retrospective series in an elderly population.

To evaluate in routine hospital practice the clinical response to ertapenem in comparison with other parenteral antibiotics in the treatment of community-acquired pneumonia (CAP), clinical records from patients with severe CAP treated with ertapenem from July 2002 to June 2006 in seven Spanish hospitals were retrospectively reviewed. Patients were classified according to the Pneumonia Severity Index (PSI). Each ertapenem-treated patient was matched with two patients in the same hospital treated with other antibiotics, according to age (difference 76 years). Comorbidities were present in 193 patients (95.5%). No differences were found in median hospital stay (7 days for ertapenem vs. 10 days for comparators, p 0.066). A slightly higher clinical response rate was obtained for ertapenem vs. comparators (88.7% vs. 77.1%; p 0.0465; OR 2.25; 95% CI 0.99-5.12), with significant differences in clinical response in patients coming from nursing homes (95.8% ertapenem vs. 63.8% comparators; p 0.0034) but not in non-institutionalized patients (85.4% ertapenem vs. 84.5% comparators; p 0.929). The higher clinical response to ertapenem vs. comparators in severe CAP was due to its significantly higher efficacy in healthcare-associated CAP in patients coming from nursing homes.

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance.

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

[Case report article: ovulation and Diane-35, current perspectives in detection and control of fertility]. / A propósito de un caso: ovulación y Diane-35, perspectivas actuales en detección y control de la fertilidad.

This is a case report article describing a patient receiving treatment for alopecia with Diane-35, in whom a recruited dominant follicle and a cumulus oophorus were detected by ultrasound. The current perspectives in the detection and control of fertility are discussed and subjected to critical analysis, with emphasis on some results concerning artificial birth control. The recognition of the ovulatory period through physiological changes, the detection of estrogeno-3-glucuronide (E3G) and the luteinizing hormone (LH) is discussed. These methods increase the possibility of understanding the detection of the fertile phase in women, are simple to use, are not invasive, do not have secondary effects, and are not abortive.

A propósito de un caso: ovulación y Diane-35, perspectivas actuales en detección y control de la fertilidad / Case report article: Ovulation and Diane-35, current prerspectives in detection and control of fertility

A propósito de un caso: Se trata de una paciente en tratamiento poralopecia con Diane-35, en la cual se detecta un folículo dominantereclutado y un cumulus oophorus diagnosticado mediante ecografía. Secomentan las perspectivas actuales en la detección y control de la fertilidadmediante análisis crítico, resaltando algunos resultados relacionadoscon el control artificial de la fertilidad, el reconocimiento del periodoovulatorio mediante la detección de cambios fisiológicos en los órganosdiana del aparato genital femenino, la detección de metabolitosestrógeno-3-glucuronido (E3G) y la hormona luteinizante (LH). Estosúltimos métodos aumentan la disponibilidad de comprender la detecciónde la fase fértil en la mujer, son sencillos de usar, no invasivos,carecen de efectos secundarios y no son abortivos

This is a case report article describing a patient receiving treatment foralopecia with Diane-35, in whom a recruited dominant follicle and acumulus oophorus were detected by ultrasound. The current perspectivesin the detection and control of fertility are discussed and subjected tocritical analysis, with emphasis on some results concerning artificialbirth control. The recognition of the ovulatory period through physiologicalchanges, the detection of estrogeno-3-glucuronide (E3G) and theluteinizing hormone (LH) is discussed. These methods increase thepossibility of understanding the detection of the fertile phase in women,are simple to use, are not invasive, do not have secondary effects, andare not abortive

DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly(ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant.

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP-/- mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by gamma-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body gamma-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP-/- cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.

In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described. We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

A eukaryotic expression vector for the study of nuclear localization signals.

We describe a new vector designed to produce beta-galactosidase fusion proteins which can be used to assess subcellular localization of target peptide fragments or proteins in eukaryotic cells. The vector was constructed in such a way as to produce the peptide of interest in fusion via a short linker of proline residues to the N terminus of the reporter protein. Efficiency of the transport machinery is optimized using this particular protein fusion construction. This vector has potential uses for readily testing putative nuclear localization sequences and identifying their crucial amino-acid residues.

Crystallization and X-ray crystallographic analysis of recombinant chicken poly(ADP-ribose) polymerase catalytic domain produced in Sf9 insect cells.

Poly (ADP-ribose) polymerase (PARP) participates in the immediate response in mammalian cells exposed to DNA-damaging agents. Recombinant baculovirus harboring the cDNA of the chicken PARP catalytic domain (40 kDa) have been used to infect Spodoptera frugiperda (Sf9) insect cells. The recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. The enzymatic properties of the recombinant domain were similar to those of the native fragment. Crystals of the purified recombinant catalytic domain were grown by vapor diffusion. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 59.2 A, b = 65.0 A, c = 96.9 A. They are suitable for X-ray analysis and diffract to 2.0 A.

Granular cell tumor of vulva on episiotomy scar.

The granular cell tumor (GCT) is a rare neoplasm derived from Schwann cells and is considered to be a benign tumor. The vulvar region is one of the common sites for these lesions. Vulvar occurrence of GCT constitutes about 7% of cases. To our knowledge, this is the first case of granular cell tumor of the vulva on an episiotomy scar. GCT should be considered in the differential diagnosis of a benign tumor of the vulva and reactive granular cell lesions.

The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA.

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a zinc-binding protein that specifically binds to a DNA strand break in a zinc-dependent manner. We describe here the cloning and expression in Escherichia coli of a cDNA fragment encoding the two putative zinc fingers (FI and FII) domain of the human poly(ADP-ribose) polymerase. Using site-directed mutagenesis, we identified the amino acids involved in metal coordination and analyzed the consequence of altering the proposed zinc-finger structures on DNA binding. Disruption of the metal binding ability of the second zinc finger, FII, dramatically reduced target DNA binding. In contrast, when the postulated Zn(II) ligands of FI were mutated, the DNA binding activity was only slightly affected. DNase I protection studies showed that the FII is involved in the specific recognition of a DNA strand break. These results demonstrate that poly(ADP-ribose) polymerase contains a type of zinc finger that differs from previously recognized classes in terms of both structure and function.

Cauliflower mosaic virus gene I product detected in a cell-wall-enriched fraction.

Gene I product of cauliflower mosaic virus was immunodetected in a cell-wall-enriched fraction from infected turnip leaves in addition to its detection in viroplasms and replication complexes. The immunoreaction was carried out with an antiserum raised against a 15 amino acid long synthetic peptide corresponding to the carboxy-terminus of potential gene I protein (P1). The presence of P1 in different subcellular fractions was investigated as a function of time during viral multiplication. At late infection times, P1 was found only in the cell-wall-enriched fraction.