Development of a Synthetic Surfactant Using a Surfactant Protein-C Peptide Analog: In Vitro Studies of Surface Physical Properties

PURPOSE: Pulmonary surfactant (PS) replacement has been the gold standard therapy for neonatal respiratory distress syndrome; however, almost all commercial PSs contain animal proteins. We prepared a synthetic PS by using a human surfactant protein (SP) analog and evaluated its in vitro properties. MATERIALS AND METHODS: A peptide sequence (CPVHLKRLLLLLLLLLLLLLLLL) of human SP-C was chosen to develop the peptide analog (SPa-C). The new synthetic SP-C PS (sSP-C PS) was synthesized from SPa-C, dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and palmitic acid. Physical properties of the sSP-C PS were evaluated by measuring the maximum and minimum surface tensions (STs), surfactant spreading, and adsorption rate. In addition, we recorded an ST-area diagram. The data obtained on sSP-C PS were subsequently compared with those of purified natural bovine surfactant (PNBS), and the commercial product, Surfacten(R). RESULTS: The sSP-C PS and Surfacten(R) were found to have maximum ST values of 32-33 mN/m, whereas that of PNBS was much lower at 19 mN/m. The minimum ST values of all three products were less than 10 mN/m. The values that were measured for the equilibrium ST of rapidly spreading sSP-C PS, Surfacten(R), and PNBS were 27, 27, and 24 mN/m, respectively. The surface adsorptions were found to be the same for all three PSs (20 mN/m). ST-area diagrams of sSP-C PS and Surfacten(R) revealed similar properties. CONCLUSION: In an in vitro experiment, the physical properties exhibited by sSP-C PS were similar to those of Surfacten(R). Further study is required to evaluate the in vivo efficacy.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

Structural and functional analysis of cathepsin S of Heterodera spp: A promising candidate for its control.

Cysteine proteinases are required for a wide range of physiological processes in all living organisms. In parasitic nematodes, they are particularly crucial for the digestion of host tissues and evasion of host immune responses. Therefore, in general, these are identified as primary targets for the control of parasitic nematodes. Herein, cathepsin S-like cysteine proteinase of Heterodera avenae (Hacp-s) has been cloned and analysed for the first time. The predicted protein is 298 amino acids long and showed significant similarity with cathepsin S of Heterodera glycines (Hgcp-s). The sequence of cathepsin S contains a signal peptide of 30 amino acids which suggests its role in extracellular functions. Multiple sequence alignment revealed the presence of ERFNIN motif and conserved catalytic residues. Three dimensional structure (3D) of Hgcp-s was modelled using homology modelling. In order to illustrate the plausible mode of interaction of cathepsin S (Hgcp-s), docking analysis was performed with E-64 cysteine proteinase inhibitor. Docking studies revealed the hydrogen bonding of E-64 with Gln153, His299 and Gly203 as well as close interaction with catalytic residues Cys159 and Asn320. Expression analysis of Hacp-s using qRT-PCR showed high expression of cathepsin S in pre parasitic J2s and female stages suggesting its significant role in both pre-parasitic and parasitic stages of the nematode life cycle.

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22.

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features — nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.

Novel mutation in MCT8 gene in a Brazilian boy with thyroid hormone resistance and severe neurologic abnormalities / Mutação nova do gene MCT8 em menino Brasileiro com resistência ao hormônio tireioidiano e neuropatia grave

O MCT8 é um transportador celular de hormônios tireoidianos, importante para sua ação e metabolização. Relatamos o caso de um menino com a nova mutação inativadora 630insG no éxon 1 do MCT8. O paciente caracterizou-se por grave comprometimento neurológico (inicialmente com hipotonia global, evoluindo com hipertonia generalizada), crescimento normal nos dois primeiros anos de vida, reduzido ganho ponderal e ausência dos sinais e sintomas típicos de hipotireoidismo. A sua avaliação sérica revelou elevação do T3, redução do T4 total e livre e TSH levemente aumentado. O tratamento com levotiroxina melhorou o perfil hormonal tireoidiano, mas não modificou o quadro clínico do paciente. Esses dados reforçam o conceito de que o papel do MCT8 é tecido-dependente: enquanto os neurônios são altamente dependentes do MCT8, o osso, o tecido adiposo, o músculo e o fígado são menos dependentes do MCT8 e, portanto, podem sofrer as consequências da exposição a níveis séricos elevados de T3.

MCT8 is a cellular transporter of thyroid hormones important in their action and metabolization. We report a male patient with the novel inactivating mutation 630insG in the coding region in exon 1 of MCT8. He was characterized clinically by severe neurologic impairment (initially with global hypotonia, later evolving with generalized hypertonia), normal growth during infancy, reduced weight gain, and absence of typical signs and symptoms of hypothyroidism, while the laboratory evaluation disclosed elevated T3, low total and free T4, and mildly elevated TSH serum levels. Treatment with levothyroxine improved thyroid hormone profile but was not able to alter the clinical picture of the patient. These data reinforce the concept that the role of MCT8 is tissue-dependent: while neurons are highly dependent on MCT8, bone tissue, adipose tissue, muscle, and liver are less dependent on MCT8 and, therefore, may suffer the consequences of the exposition to high serum T3 levels.

Building multiple sequence alignments with a flavor of HSSP alignments

Homology-derived secondary structure of proteins (HSSP) is a well-known database of multiple sequence alignments (MSAs) which merges information of protein sequences and their three-dimensional structures. It is available for all proteins whose structure is deposited in the PDB. It is also used by STING and (Java)Protein Dossier to calculate and present relative entropy as a measure of the degree of conservation for each residue of proteins whose structure has been solved and deposited in the PDB. However, if the STING and (Java)Protein Dossier are to provide support for analysis of protein structures modeled in computers or being experimentally solved but not yet deposited in the PDB, then we need a new method for building alignments having a flavor of HSSP alignments (myMSAr). The present study describes a new method and its corresponding databank (SH2QS--database of sequences homologue to the query [structure-having] sequence). Our main interest in making myMSAr was to measure the degree of residue conservation for a given query sequence, regardless of whether it has a corresponding structure deposited in the PDB. In this study, we compare the measurement of residue conservation provided by corresponding alignments produced by HSSP and SH2QS. As a case study, we also present two biologically relevant examples, the first one highlighting the equivalence of analysis of the degree of residue conservation by using HSSP or SH2QS alignments, and the second one presenting the degree of residue conservation for a structure modeled in a computer, which , as a consequence, does not have an alignment reported by HSSP.

Molecular characterization of ABC transporter-encoding genes in Aspergillus nidulans

As a preliminary step towards characterizing genes encoding ATP-binding cassette (ABC) transporters that confer pleiotropic drug resistance in Aspergillus, we used a PCR-based approach to isolate four DNA fragments corresponding to different ABC type transporter genes. DNA sequencing and Southern blot analysis confirmed that they were distinct genes, which were designated abcA-D. One of these genes, abcD, was cloned and characterized. It was found to have a predicted 1,452-amino acid translation product with a calculated molecular mass of 147,467 kDa. The abcD gene specifies a single transcript of approximately 5.0 kb; there was a two- to six-fold enhancement of mRNA levels following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide

Diverse VacA Allelic Types of Helicobacter pylori in Korea and Clinical Correlation

Helicobacter pylori has a diversity of vacA allelic types. The purpose of this study was to correlate the vacA status and the clinical outcome. After constructing specific primers for the vacA signal sequence, H. pylori-positive antral biopsy specimens were examined for the vacA status in 25 gastric ulcers, 31 duodenal ulcers, 22 gastric cancers, 42 chronic gastritis, and 8 gastroduodenal ulcers. The relationship between the vacA allele and the clinical disease was examined. The vacA genotype s1c/m1 is predominant in Korea (71/128, 55.5%). Other strains including s1b or s2 were not found in this study. s1c/m1 was more prominent in duodenal ulcers, than in gastric ulcers (p=0.041) and cancer (p=0.029). Seven out of 8 patients with gastric and coexistent duodenal ulcers had the s1c/m1 allele. No statistical differences in the positive rates of the s1a/m1, s1a/m2, and s1c/m2 alleles among the disease groups were found. In conclusion, s1c/m1 is the main vacA allele in Korea and it is particularly associated with duodenal ulcers.

A polymerase chain reaction-based assay to identify genotype F of hepatitis B virus

We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4 percent) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America

Identification and purification of IgE-reactive proteins in German cockroach extract

Cockroaches have been implicated as a cause of respiratory allergy in urban areas worldwide. IgE-reactive German cockroach proteins were identified with molecular weights (MWs) of 90, 66, 50, 43 and 36 KD by immunoblot analysis in both immune BALB/c mice and sensitized humans. Prominent IgE-reactive proteins were purified using FPLC by ion-exchange chromatography, gel filtration and hydrophobic chromatography. The N-terminal amino acid sequence of a purified protein with a MW of 66 KD on SDS-PAGE was Val-Thr-Leu-Lys-Lys(Val)-Met-Ile-Lys-Thr-Phe-Tyr. No homologous protein was found through a search of GenBank that indicated a novel IgE-reactive protein in German cockroach extract. Another purified protein with a MW of 36 KD reacted strongly with a monoclonal antibody against Bla g 2.

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.

Secuenciacion directa de un producto amplificado de una muestra de suero / Direct sequence of an amplified product from a serum sample

Se reporta la secuencia nucleotida y aminoacidica de una zona de gran variabilidad en el genoma del virus dengue 2 a partir del ARN del virus original sin pase en ningun sistema de aislamiento y se compara con la primera cepa de dengue 2 aislada durante la epidemia de 1981 con 4 pases en raton lactante. Los resultados demuestran que la secuencia nucleotidica del suero y de la cepa A15 son iguales

Pyruvate: ferredoxin oxidoreductase from Entamoeba histolytica recognized by a monoclonal antibody.

A mouse monoclonal antibody, Eh208C2-2 MAb, raised against whole cell antigens of Entamoeba histolytica trophozoites of the pathogenic strain HM-1: IMSS and polyclonal antisera (PAb) against membrane antigens of E. histolytica trophozoites of strain HTH-56: MUTM were screened against a cDNA library of the pathogenic strain, SFL3. The monoconal antibody detected many phage plaques expressing an E. histolytica protein. The DNA sequence encoding the protein was approximately 55% identical, over 1,100bp, to Trichomonas vaginalis pyruvate: ferredoxin oxidoreductase (PFOR) and pyruvate: flavodoxin oxidoreductase from Klebsiella pneumoniae, Anabaena variabilis and Enterobacter agglomerans. Two of seven clones detected by mouse polyclonal antisera also encoded this protein. Two others encoded Entamoeba Hsp70, another encoded Entamoeba alkyl-hydroperoxide reductase and the remaining two were unidentified sequences. Entamoeba PFOR is an abundant, antigenic protein which may be a useful target for the development of protective host immune responses against invasive amebiasis.

Heterogeneidad genomica de virus de la hepatitis B, genotipo A, circulantes en el area metropolitana de Buenos Aires, Argentina / Genomic heterogenity of virus B Hepatitis, genotype A circulating in Buenos Aires, metropolitan area, Argentine

Se purificaron partículas de Dane, a partir de sueros de pacientes virémicos del area metropolitana de Buenos Aires (Argentina) y se extrajo HBV DNA. El HBV DNA fué clonado en el vector pUC18, amplificado en Escherichia coli DH5 alpha F'. Los plasmidos fueron aislados y el contenido de los insertos de HBV DNA se analizó por tratamiento con diversas enzimas de restricción para estabelecer las características genómicas clonadas. Se seleccionaron tres plasmidos y sus estructuras fueron determinadas, identificandolos como: pHB4, pHB7 y pHB20. Los insertos presentes en cada uno fueron secuenciados y el resultado incorporado a la base de datos Gen Bank, con los siguientes números de código: PHB4P3= U33188; PHB4P5= U33189; PHB7P3=U33190; PHB7P5=U33191 y PHB20=U33187. Todos los insertos pertenencen a genotipos A, pHB4 y pHB20 tienen una estrecha homología, que comparten con el L 13994, de circulación americana. pHB7 presenta una marcada diferencia con los clones pHB4 y pHB20 y una discreta relación con el agente m57663, aislando originalmente en Filipinas. pHB4 muestra una mutación en el nucleótico T3182(Leu en la región preS1, que cambia Pro. por Leu. Esta mutación est ausente en 125 secuencias estudiadas, que mantienen por lo menos un 65 por ciento de homología con los clones aislados en el area metropolitana de Buenos Aires. Esta selección se efectuó en el banco National Center for Biotechnology information (NCBI) utilizando el algoritmo Blast. El an lisis de estos clones no reveló la presencia de mutantes del tipo e o de escape HBV DNA, con este tipo de variantes han sido descriptas recientemente en esta area geografica por López y col. (Ref.7). Si bien los genotipos aislados pertencen al tipo A, es de importancia tener en cuenta la manifesta divergencia de por lo menos uno de los clones estudiados.

The sound of the DNA language

A new method is described for the study of DNA language employing the communicative strength of music. An algorithm was created to ®translate® codons into musical notes. The analysis of the musical transcriptions of DNA sequences suggests the presence of some structural features of DNA language hidden in human and mouse interferon alpha 1 genes

Analysis of Japanese encephalitis (JE) virus genome and implications for recombinant JE vaccine.

From the information of nucleotide sequences and deduced amino acid sequences of flaviviruses including JEV, we can postulate processing mechanisms of a polyprotein translated from single long open reading frame of the genome and mechanisms of construction of antigenic structures of structural proteins with biologically active forms after these proteins are translated. The results of comparative analysis of amino acid sequences among flaviviruses and epitope analysis on the E proteins which are the most important antigens for protective immunity suggest that the E protein of flaviviruses may have a similar structure closely related to each other. PrM and E proteins which had predictable signal sequences upstream on the N terminals were expressed with antigenically active form and molecular size the same as the authentic ones by the recombinant viruses. However, the recombinant viruses which had no such signal sequence expressed unprocessed proteins with antigenically denatured forms. These results suggest that normal proteolytic processing is needed to construct biologically active structures of JEV structural proteins. The E proteins which were expressed by the recombinant viruses as antigenically active form could elicit nutralizing and HI antibodies in animals and protective immunity in mice. The recombinant vaccinia viruses which express the E protein could induce strong immunologic memory against the E protein in mice. These results indicate that the development of a new type of vaccine against JEV will become possible in future.

Overview of flavivirus molecular biology and future vaccine development via recombinant DNA.

Studies in many laboratories over the last several years have elucidated the structures of several different flavivirus genomes. Conserved features include the production of at least 10 different virus encoded proteins from a single long open reading frame by a combination of host and virus-encoded proteases. The established gene order is 5'-C- prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS 5-3' and these proteins exhibit varying degrees of homology in comparisons among flaviviruses. Conserved RNA sequences and structures have also been identified for the mosquito-borne flaviviruses but are absent in sequenced tick-bone viruses. Relevant to the development of efficacious flavivirus vaccines, studies aimed at defining the antigenic determinants necessary for eliciting protective immunity have focused primarily on the structural proteins, in particular the E protein, as well as the nonstructural secreted glycoprotein, NS1. Other work, which has led to the derivation of live-attenuated flavivirus strains, should eventually allow the genetic determinants of flavivirus attenuation and pathogenesis to be understood at the molecular level. The successful recovery infectious flaviviruses from cloned cDNA raises the possibility of manipulating these viral genomes as cDNA to construct or propagate candidate live-attenuated vaccine strains. Several applications of this technology are discussed.