The effects of CO2 pressure and pH on the Suzuki coupling of basic nitrogen containing substrates.

The Suzuki coupling reaction of basic nitrogen containing substrates (2-bromo- and 2-chloro-4-aminopyridine, and 2-bromo and 2-chloropyridine) with phenylboronic acid using Pd(TPP)2Cl2/K3PO4 in acetonitrile-water biphasic solvent systems under a CO2 or a N2 atmosphere is discussed. It was observed that 2-halo-4-aminopyridine produced quantitative yields of coupled products under a CO2 atmosphere while the yields for the 2-halopyridines were poor. In contrast, the yields of coupled products for the 2-halopyridines substrates were quantitative under a N2 atmosphere while only poor yields were realized for the 2-halo-4-aminopyridines under the same conditions. Evidence is presented which suggests that the presence of CO2 alters the pH of the aqueous phase of the reaction system and the accompanying efficiency of the coupling process. Using a series of buffers to adjust the pH of the aqueous phase, the pH dependence associated with the efficiency of the coupling process is illustrated.

Endemic pemphigus foliaceus in the Peruvian Amazon.

BACKGROUND: Endemic pemphigus foliaceus (EPF) is an organ-specific blistering disease of the epidermis characterized by the presence of IgG autoantibodies, specifically desmoglein (Dsg)1. This condition has been reported particularly in Brazil, Colombia, Tunisia and Peru. AIM: To characterize the humoral response against Dsg1 and Dsg3 autoantibodies of patients with EPF from the Peruvian Amazon region. METHODS: Blood samples were collected from 16 patients with a clinical diagnosis of EPF, and tested using indirect immunofluorescence (IIF), immunoprecipitation and ELISA (for IgG and its subclasses against Dsg1 and IgG against Dsg3). RESULTS: Autoantibodies against the intercellular spaces were detected by IIF in 82.5% and 87.5% of patients, using normal human skin and monkey oesophagus, respectively. Sera from all patients immunoprecipitated recombinant Dsg1, and three serum samples immunoprecipitated recombinant Dsg3 (6.25%). Using ELISA, anti-Dsg1 antibodies were detected in 13 patients (81.25%), and both IgG1 and IgG2 antibodies against Dsg1 in 12 patients (75%). All patients were positive for IgG4 autoantibodies, and only one patient was positive for IgG3 autoantibodies (6.25%). Anti-Dsg3 antibodies were detected in five patients (31.25%). CONCLUSIONS: EPF from Peru shares epidemiological, clinical and immunological characteristics with other forms of EPF that have been described in South America.

Effectiveness and safety of the schedules of short and long term treatment for tuberculous meningoencephalitis at two hospitals of Lima--Peru.

INTRODUCTION: To compare the effectiveness and safety of short term 6 month-treatment and long term 12 month-treatment schedules for meningoencephalitis due to tuberculosis in two hospitals from Lima-Peru. METHODS: Comparative, retrospective and observational study. The patients were divided in two groups: Group 1: long term 12 month-treatment with isoniazid, rifampin, pyrazinamide, and ethambutol for the first 2 months; then isoniazid and rifampin for 10 months. Group 2: short term 6 month-treatment with isoniazid and rifampin, pyrazinamide and ethambutol for the first 2 months; then isoniazid and rifampin for 4 months. Clinical records, effectiveness, treatment failure, treatment side effects, mortality and late consequences after treatment were reviewed. RESULTS: Twenty-six patients with meningoencephalitis level I were included, 10 received the long term schedule and 16 the short term schedule treatment. From 51 patients with meningoencephalitis level II, 27 received the long term schedule and 24 the short term schedule treatment and of 31 patients with meningoencephalitis level III, 18 received the long term schedule treatment and 13 the short term schedule treatment. There was no statistically significant differences among levels I, II and III when effectiveness of short and long term schedule was evaluated. Moreover, there was no statistically significant difference in the frequency of treatment failure, treatment side effects, mortality and late consequences among groups. CONCLUSIONS: Long term 12 month-treatment and short term 6 month-treatment had similar effectiveness and safety in the treatment of meningoenchephalitis due to tuberculosis in HIV negative patients.

Recognition and management of dermatologic complications from interventional radiology procedures.

A variety of dermatologic complications can occur after interventional radiology procedures, including fluoroscopy-induced radiation dermatitis, thermal skin injury from tumor ablation, non-target embolization to the skin, allergic reactions related to interventional radiology procedures, and dermatitis and infections at catheter sites. Yet, interventional radiologists typically lack training in dermatology. This review focuses on recognition of dermatologic complications and introduces basic principles for management of these complications. By taking a more active role in the diagnosis, management, and follow-up of dermatologic complications, interventional radiologists can improve the care for patients suffering iatrogenic skin inury.

Noninfectious dermatological diseases associated with chronic exposure to mine tailings in a Peruvian district.

BACKGROUND: Mine tailings are metallic wastes which are deposited in the environment due to mining activity. Long-term exposure to these metals is harmful to human health. OBJECTIVE: To determine if chronic exposure to mine tailings constitutes a risk factor for the development of dermatological diseases in the district of San Mateo de Huanchor (Lima, Peru). METHODS: An observational case-control study was carried out in the communities of Mayoc, Daza and Tamboraque (exposed to mine tailings, case group) located in the district of San Mateo de Huanchor, and also in the communities of Choccna and Caruya (not exposed to mine tailings, control group) located in the same district. Out of 230 adults, 121 were exposed and 109 were not exposed to mine tailings and out of 135 children, 71 were exposed and 64 were not exposed to mine tailings. RESULTS: In the adult group, 71% of the exposed cases had some noninfectious dermatological disease while in the nonexposed group the frequency was 34% [P < 0.001; odds ratio (OR) 5.40; 95% confidence interval (CI) 3.02-9.68]. A statistically significant difference between groups was found for arsenical dermatitis, nonpruritic papulovesicular eruption, atopic dermatitis, contact dermatitis, seborrhoeic dermatitis and xerosis. In the paediatric population, 71 exposed and 64 nonexposed children were evaluated. Sixty-nine per cent of the exposed group had some noninfectious dermatological disease vs. 30% in the nonexposed group (P < 0.001; OR 6.00; 95% CI 2.71-13.31). A statistically significant difference between groups was found for xerosis and atopic dermatitis. CONCLUSION: Chronic exposure to mine tailings represents a risk factor for development of noninfectious dermatological diseases in both adults and children.

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

Influence of Si addition on the microstructure and mechanical properties of Ti-35Nb alloy for applications in orthopedic implants.

In the development of new materials for orthopedic implants, special attention has been given to Ti alloys that show biocompatible alloy elements and that are capable of reducing the elastic modulus. Accordingly, Ti-Nb-Si alloys show great potential for application. Thus, this is a study on the microstructures and properties of Ti-35Nb-xSi alloys (x=0, 0.15, 0.35 and 0.55) (wt%) which were thermally treated and cooled under the following conditions: furnace cooling (FC), air cooling (AC), and water quenching (WQ). The results showed that Si addition is effective to reduce the density of omega precipitates making beta more stable, and to produce grain refinement. Silicides, referred as (Ti,Nb)3Si, were formed for alloys containing 0.55% Si, and its formation presumably occurred during the heating at 1000°C. In all cooling conditions, the hardness values increased with the increasing of Si content, as a result from the strong Si solid solution strengthening effect, while the elastic modulus underwent a continuous reduction due to the reduction of omega precipitates in beta matrix. Lower elastic moduli were observed in water-quenched alloys, which concentration of 0.15% Si was more effective in their reduction, with value around 65 GPa. Regarding Ti-35Nb-xSi alloys (x=0, 0.15 and 0.35), the "double yield point" phenomenon, which is typical of alloys with shape memory effect, was observed. The increase in Si concentration also produced an increase from 382 MPa to 540 MPa in the alloys' mechanical strength. Ti-35Nb-0.55Si alloy, however, showed brittle mechanical behavior which was related to the presence of silicides at the grain boundary.

Nutritional management of thoracic duct fistulas. A comparative study of parenteral versus enteral nutrition.

Thoracic duct fistulas represent one of the serious technical complications of head and neck surgery, and reoperation for control of the leakage involves considerable morbidity and mortality. In an attempt to define the possibilities of both enteral and parenteral nutrition in the treatment of this problem, two comparable groups of patients were given, respectively, one or the other of these modalities. Significant advantages for parenteral nutrition could be demonstrated regarding duration of therapy (p less than 0.05), closure rate (p less than 0.05), and nutritional response (p less than 0.05). These results strongly recommended the utilization of parenteral nutrition in the primary therapy of thoracic duct fistulas.

Osteogenesis imperfecta and coronary artery surgery. A case report.

A new case of Osteogenesis Imperfecta (OI) suffering ischemic heart disease is reported. The patient was successfully operated on in our Institution and the bibliographic search showed only another case of such an association of diseases successfully treated by surgery. This patient proves that coronary artery surgery procedures are possible when OI complicates the cardiac ischemic syndrome.

Floristic analysis and dispersal syndromes of woody species of the Serra de Maracaju, Mato Grosso do Sul, Brazil.

This paper presents a floristic survey of the wood component of cerrado s.s. (eastern face) and gallery forest (western face) areas carried out in the southern part of the Serra de Maracaju. The dispersal syndromes and floristic relations of this portion of the Serra were compared to those of other formations from different regions of Brazil. Between October 2007 and September 2008, monthly collections identified 144 species. As for dispersal syndromes, zoochory prevails, followed by autochory and anemochory. The gallery forest is a heterogeneous formation that shelters floristic elements shared with the Atlantic rain forest, the Amazonian forest, semi-deciduous seasonal forests and dry forests of the Pantanal. Low similarity between the two areas analyzed makes this region unique and suggests that the Serra de Maracaju is greatly influenced by the semi-deciduous seasonal forests of the southeastern region.

Infecciones bacterianas en pacientes con Pie Diabético. Hospital Regional de Ciudad del Este, Paraguay. Año 2015 / Bacterial infections in patients with Diabetic Foot. Regional Hospital of Ciudad del Este, Paraguay. Year 2015

Infecciones bacterianas en pacientes con Pie Diabético. Hospital Regional de Ciudad del Este, Paraguay. Año 2015 RESUMEN La Organización Mundial de la Salud define el pié diabético como una "situación de infección, ulceración o también destrucción de los tejidos profundos de los pies, asociada a anormalidades neurológicas y varios grados de enfermedad vascular periférica en los miembros inferiores de pacientes con Diabetes Mellitus. El objetivo del trabajo fue identificar la prevalencia de microorganismos que causaron infecciones en pacientes diagnosticados con pie diabético atendidos en el Hospital Regional de Ciudad del Este, Paraguay, durante el año 2015. Estudio descriptivo, transversal y retrospectivo. El muestreo fue no probabilístico, por conveniencia, y se incluyeron 115 muestras correspondientes a 94 pacientes. Fue utilizada la base de datos del Laboratorio de Bacteriología del Hospital Regional de Ciudad del Este. De los 94 pacientes con pie diabético, 52% fueron hombres. El 25% de las infecciones se produjeron en pacientes con edades comprendidas entre 51 a 60 años. Fueron aislados 21 microorganismos diferentes en las 115 muestras. El 80% (75) fueron monomicrobianos, y 20% (40) polimicrobianos. Entre los Gram positivos, el microorganismo más frecuentemente aislado fue el Staphylococcus aureus 19% (22) y el Enterococcus spp. 6% (6) y entre los Gram negativos fueron la Klebsiella pneumoniae13% (16) y Acinetobacter spp. 12% (14). Los resultados de las pruebas de sensibilidad antimicrobiana demostraron que el 100% de las cepas de S. aureus fueron resistentes a la Oxacilina y alta resistencia de las cepas de K. pneumoniae a las Cefalosporinas. Las cepas de Acinetobacter spp. fueron 100% resistentes a las cefalosporinas y Piperacilina. Los microorganismos aislados y el perfil de resistencia antimicrobiana que los mismos presentan, coinciden con la bibliografía, y es muy importante implementar programas de prevención a esta patología a fin de evitar amputaciones en este tipo de pacientes. Palabras claves: bacterias; pie; diabético; hospital; Paraguay.

The World Health Organization defines the diabetic foot as a "situation of infection, ulceration or destruction of the deep tissues of the feet. It is associated with neurological abnormalities and various degrees of peripheral vascular disease in the lower limbs of patients with Diabetes Mellitus". The objective of the research was to identify the prevalence of microorganisms which caused infections in patients diagnosed with diabetic foot treated at the Regional Hospital of Ciudad del Este, Paraguay, during the year 2015. It is a descriptive, crosssectional and retrospective study. The sampling was nonprobabilistic, for convenience, and 115 samples were included corresponding to 94 patients. The database of the Department of Bacteriology of the Regional Hospital of Ciudad del Este was used. Of the 94 patients with diagnosed diabetic foot, 52% were men. 25% of the infections occurred in patients with ages ranging from 51 to 60 years. 21 microorganisms were isolated in the 115 different samples. 80% (75) were monomicrobial, and 20% (40) polymicrobial. The most isolated bacterium in this group of bacteria was the Staphylococcus aureus (19%). Among the Gram positive bacilli, the most frequently isolated microorganism was Staphylococcus aureus, 19% (22) and Enterococcus spp. 6% (6) and among Gram negatives: Klebsiella pneumoniae 13% (16) and Acinetobacter spp. 12% (14). The results of the sensitivity tests demonstrated that 100% of the strains of S. aureus were resistant to Oxacillin and 100% of the strains of K. pneumoniae showed resistance to cefhems. 100% of the strains of Acinetobacter spp were resistant to Cefotaxime and Piperacillin. The isolated microorganisms and the antimicrobial resistance profile they present coincide with the literature, and it is of most importance to implement prevention programs regarding this pathology in order to avoid amputations in this type of patients. Key words: bacteria; foot; diabetic; h Palabras claves: bacterias; pie; diabético; hospital; Paraguay.

[Convulsive status epilepticus: clinico-epidemiologic characteristics and risk factors in Peru]. / Estado epiléptico convulsivo: características clínico-epidemiológicas y factores de riesgo en Perú

INTRODUCTION: Convulsive status epilepticus constitutes a medical emergency that requires a fast and aggressive management with the objective to prevent neuronal damage and systemic complications. The objective is to determine clinico-epidemiologic characteristics and risk factors associated with convulsive status epilepticus in adults attended in a National Hospital from Lima-Peru in a period of four years. METHODS: Case-control study. Cases were constituted by patients admitted for convulsive status epilepticus in the Adult Emergency Service of National Hospital Dos de Mayo between January 2003 and December 2007. Controls were patients with diagnoses of epilepsy that received treatment in outpatient service of neurology, couplet by age and sex with the group of cases. Medical histories were reviewed and patients were interviewed obtaining clinical, epidemiologic and possible risk factors to convulsive status epilepticus that were registered in an instrument of data recorded. RESULTS: 41 cases of convulsive status epilepticus were presented. 68.3% were male, 28.6% had age between 20 and 29 years old and 15.5% resided in endemic areas of neurocysticercosis. The more frequent aetiologies were remote symptomatic secondary crisis to cranio encephalic trauma and neurocysticercosis and idiopathic; 26.8% showed some intercurrent infection; while, mortality was of 7.3%. Factors associated with a convulsive status epilepticus were the abrupt interruption or suspension of drugs used for the control of convulsions (p=0.038), chronic intake of alcohol (p=0.030) and irregular antiepileptic treatment (p=0.006). CONCLUSIONS: Aetiologies more frequent in the hospital studied from Lima-Peru are remote symptomatic secondary crisis to cranio encephalic trauma, neurocysticercosis and idiopathic. The irregular antiepilepticus treatment constitutes a risk factor to convulsive status epilepticus.

[Etiology and risk factors for a first episode of cerebral isquemia in young adults]. / Etiología y factores de riesgo para un primer episodio de isquemia cerebral en adultos jóvenes.

INTRODUCTION: To determinate the etiology and risk factors for a first episode of cerebral ischemia in young adults at three hospitals of Lima and Callao-Peru. METHODS: Multicentric study carried out at three national hospitals in Lima. The sample included 30 patients with a first episode of stroke and 60 controls matched by age and sex 2:1 with the patients. Serum biochemistry studies, EKGs and echocardiograms were done. Etiologies were classified based on the classification of Baltimore-Washington Cooperative Young Stroke Study. RESULTS: The most frequent etiologies were cardiac embolism and atherosclerotic valvular heart disease, which were 30% of the cases (9 patients) each one. Hypertriglyceridemia (p=0.014), valvular heart disease (p=0.001) and hormonal contraception/replacement therapy (p=0.002) were independent risk factors for a first episode of cerebral ischemia in peruvian young adults. Motor deficiency was the most frequent presentation (50.0%). Intracraneal hypertension and urinary tract infection were the most frequent complications during acute ischemia and mortality was raised up to 10%. CONCLUSIONS: The hypertriglyceridemia, valvular heart disease and the use of oral contraceptives are independent risk factors for a first episode of ischemia in young adults from three hospitals of Lima and Callao. The most frequent etiologies were cardiac embolism and atheroesclerotic valvular heart disease.

Remediation of lignin and its derivatives from pulp and paper industry wastewater by the combination of chemical precipitation and ozonation.

In the present work the degradation of the lignin and its derivatives in the residual water of a paper industry by simple ozonation was investigated. The remediation of lignin was realized using the combination of the pre-treatment with chemical precipitation, using concentrated sulfuric acid (97.1%) at the pH 1 and 3, and of the simple ozonation of the filtered residual water at the pH 1, 3, 8 and 12. Since the high residues content (the initial chemical oxygen demand (COD) is 70,000 mg/L) in the experiments the diluted samples (1:10) were used. The previous precipitation has showed a significant effect on the reduction of the COD (77%) and color (96.1%). The sludge precipitated contents sulfolignin, which in the reaction with sulfuric acid was formed. In ozonation of the filtered residual water during 25 min at the pH 1, 3, 8 and 12 the follows by-products were formed: fumaric, maleic, malonic and formic acids. The biodegradability of the treated water in ozonation increases up 0.067-0.29. The effect of the precipitation and the ozonation conditions on the decolorization kinetics was evaluated.

Glutamate and kainate effects on the noradrenergic neurons innervating the rat vas deferens.

The effects of glutamate on the noradrenergic innervation of the rat vas deferens have been investigated. Administration of a single dose of kainate (50 micrograms) to the peripheral noradrenergic ganglia innervating the vas deferens induced a time-dependent decrease of norepinephrine in the organ; after 10 days the norepinephrine concentration had fallen to 35% of control values. This effect was accompanied by a 70% decrease in the potassium-induced release of recently incorporated 3H-norepinephrine. Concomitantly, postsynaptic hypersensitivity to both norepinephrine and dopamine appeared. The finding that adrenergic ganglia possess high-affinity glutamate binding sites suggests that the effect of kainate may be ascribed to glutamate receptors present on the perikarya of the noradrenergic neurons. It is concluded that noradrenergic neurons of the vas deferens are under glutamatergic control and that this might be important in the motor control of the organ.

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.

Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex.

The yeast recombination and repair proteins Rad1 and Rad10 associate with a 1:1 stoichiometry to form a stable complex with a relative molecular mass of 190 kDa. This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically, also cleaves supercoiled duplex DNA molecules. In this reaction, supercoiled (form I) molecules are rapidly converted to nicked, relaxed (form II) molecules, presumably as a result of nicking at transient single-stranded regions in the supercoiled DNA. At high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules. The Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity. The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of the RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incision events of nucleotide excision repair and the removal of nonhomologous 3' single strands during intrachromosomal recombination between repeated sequences. In these pathways, the specificity and reactivity of the Rad1/Rad10 endonuclease will probably be modulated by further protein-protein interactions.

Biochemical and genetic characterization of the DNA ligase encoded by Saccharomyces cerevisiae open reading frame YOR005c, a homolog of mammalian DNA ligase IV.

Here we demonstrate that the Saccharomyces cerevisiae DNA ligase activity, which we previously designated DNA ligase II, is encoded by the genomic DNA sequence YOR005c. Based on its homology with mammalian LIG4, this yeast gene has been named DNL4 and the enzyme activity renamed Dnl4. In agreement with others, we find that DNL4 is not required for vegetative growth but is involved in the repair of DNA double-strand breaks by non-homologous end joining. In contrast to a previous report, we find that a dnl4 null mutation has no effect on sporulation efficiency, indicating that Dnl4 is not required for proper meiotic chromosome behavior or subsequent ascosporogenesis in yeast. Disruption of the DNL4 gene in one strain, M1-2B, results in temperature-sensitive vegetative growth. At the restrictive temperature, mutant cells progressively lose viability and accumulate small, nucleated and non-dividing daughter cells which remain attached to the mother cell. This novel temperature-sensitive phenotype is complemented by retransformation with a plasmid-borne DNL4 gene. Thus, we conclude that the abnormal growth of the dnl4 mutant strain is a synthetic phenotype resulting from Dnl4 deficiency in combination with undetermined genetic factors in the M1-2B strain background.