Short communication: Homologous expression of recombinant and native thurincin H in an engineered natural producer.

The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

Shelf-life evaluation of natural antimicrobials for Concord and Niagara grape juices.

This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10(2) and 10(4) CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21°C by yeast enumeration and measurement of turbidity, pH, and °Brix. Juices were deemed spoiled when yeast counts exceeded 10(6) CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, pH, and °Brix. Spoiled juices had lower pH and °Brix values and higher turbidity due to microbial activity and increased in microbial levels.

Prediction of the oversulphated chondroitin sulphate contamination of unfractionated heparin by ATR-IR spectrophotometry.

The detection of a contamination of heparin with oversulphated chondroitin sulphate (OSCS) was first analysed in an unfractionated heparin batch supplied to the US API-market in April 2006. OSCS is a semi-synthetic derivative of the natural occuring glycosaminoglycan chondroitin sulphate. Moreover some spectroscopic characteristics of the substance overlap with those of heparin, so that the infrared (IR) spectra are visually difficult to distinguish whereas (1)H-NMR (Nuclear Magnetic Resonance) spectroscopy or capillary electrophoresis (CE) provides identification by a simple visual inspection of either the spectrum or the electropherogram respectively. However, applying special tools of Multivariate Data Analysis (MVA) to the IR spectra an identification of the contaminated samples is possible. In detail a rapid Attenuation Total Reflectance-Infrared (ATR-IR) measurement was selected, which does not require any sample preparation. The result (contaminated or not contaminated) is predicted within a few minutes. A method transfer to mobile ATR-IR spectrometers seems to be possible. The analysis is based on the fact that the fingerprint of the OSCS IR spectrum (1st derivative) complies with a theoretically calculated principal component in the MVA.

Histerectomía total por videolaparoscopia: experiencia en Antofagasta / Total hysterectomy by videolaparoscopy: experience at Antofagasta

Se informa sobre 96 casos de histerectomía total por vía laparoscópica, efectuadas con electrocoagulación bipolar, y corte con tijera y electrobisturí monopolar. La media de edad fue de 45 años, de paridad 2,87 y de aborto 0,78. Setenta y seis pacientes tenían antecedente de cirugía abdominal previa. El tiempo quirúrgico medio fue de 134,48 minutos (rango de 70 minutos a 4 horas). Hubo lesión de uréter en 2 pacientes y en 4 casos se necesitó de transfusión por sangrado intraoperatorio. Se convirtieron a laparotomía 7 casos. En 59 pacientes se practicaron otros procedimientos. El tiempo promedio de hospitalización fue de 51,9 horas. El peso promedio del útero fue de 300 g con mínimo de 105 y máximo de 620 g

Antimalarial activity of 1,4-epidioxy-bisabola-2,12-diene derivatives.

1,4-Epidioxy-bisabola-2,12-diene (3) and aromatic hydroperoxides (4, 5) were prepared by photoxidation of gamma-curcumene (1). Reduction and esterification of 6 and 7 afforded compounds 9 to 10. All compounds were tested in vitro for antimalarial activity. The activity could not be increased significantly, compared with 3. The most active compounds, 3 and 9, did not show in vivo antimalarial activity in mice.

Allergenic sesquiterpene lactones from Eupatorium cannabinum L. and Kaunia rufescens (Lund ex de Candolle).

From Eupatorium cannabinum L., a hitherto unknown alpha-methylene-gamma-butyrolactone, 3 beta-peroxyeucannabinolide, was isolated. This compound and eupatoriopicrin from the same plant showed a weak sensitizing capacity in guinea pigs. 2-oxoludartin and dehydroleucodin, isolated from Kaunia rufescens (syn. Eupatorium rufescens), were strong sensitizers in the same sensitizarian procedure.

Sesquiterpene peroxides from Senecio selloi and Eupatorium rufescens.

Two bisabolane-type sesquiterpenes, zingiberene-3,6-beta-endoperoxide (1) and zingiberene-3,6-alpha-endoperoxide (2) have been isolated from the aerial parts of Senecio selloi and Eupatorium rufescens. Their structures were determined by spectroscopic methods. Both compounds show schizonticidal activity against Plasmodium falciparum (EC50 = 10 microg/ml).

Detection of apoptosis in KG-1a leukemic cells treated with investigational drugs.

Four investigational drugs, p-benzoquinone, primine, miconidine acetate, and artesunate (dihydroqinghaosusuccinate), with growth inhibitory activity against flagellatae (e.g. trypanosoma, leptomonas, plasmodium) were investigated for their capability to induce programmed cell death (apoptosis) in human KG-1a leukemic cells. The results were compared with those of three well established cytostatic agents (cisplatin, daunorubicin, cytosine-arabinoside) and ionizing radiation. The antitumor activity of the drugs was validated by a cellular growth inhibition assay. The depletion of glutathione by these four investigational drugs favours the hypothesis that formation of free radicals and subsequent DNA strand breaks may be critical mechanisms of action and that the glutathione redox cycle is involved in detoxification of these reactive molecules.

Ablación endometrial histeroscópica: experiencia en Antofagasta / Hysteroscopic endometrial ablation: experience at Antofagasta

Se analizan 52 casos de ablación endometrial histeroscópica, realizadas entre marzo de 1992 a diciembre de 1995, procedimientos que se efectuaron con resectoscopio y asa diatérmica. El signo común de estas pacientes era genitorragia refractaria a tratamientos médicos. El seguimiento postcirugía es de 20,3 meses promedio, el 76,9 por ciento de ella están sin problemas, el 19,2 por ciento (10 casos), requirieron otra cirugía por recurrencia de sus síntomas, 8 histeroctomías

Correlación sonográfico-clínica en polimalformado / Sonographic-clinical correlation in polymalformated

Se presenta el caso clínico de paciente de 40 años controlada en nuestra Unidad de Alto Riesgo Obstétrico, desde las 33 semanas. enviada desde atención primaria por edad gestacional dudosa al encontrarse discordancia entre altura del fondo y edad gestacional estimada por la fecha de última regla. Se exponen hallazgos ecográficos y su correlación con el exámen del recién nacido, enfatizándo acerca de la utilidad de los llamados marcadores ecográficos de cromosomopatías

Linalool and cineole type glucosides from Cunila spicata.

The leaves of Cunila spicata yielded a monoterpenetriol and six glycosidic terpenoids derived from linalool, hydroxylated linalool and 1,8-cineole: 3,7-dimethyl-oct-1-ene-3,6,7-triol, linalool-O-beta-D-glucopyranoside, 3,7 dimethyl-octa-1,6-diene-3,8-diol-3-O-beta-D-glucopyranoside as well as 3,7-dimethyl-octa-1,5-diene-3,7-diol-3-O-beta-D-glucopyranoside, 3,7-dimethyl-octa-1,7-diene-3,6-diol-7-O-beta-D-glucopyranoside, 3,7- dimethyl-oct-1-ene-3,6,7-triol-6-O-beta-D-glucopyranoside and (1S,4R,6R)-1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-O-beta- glucopyranoside. The structures of the glucosides were established by chemical and spectroscopic methods especially high field NMR techniques.

Higroma quístico cervical / Cervical cystic hygroma

Se presenta un caso clínico de diagnóstico antenatal ultrasonográfico de higroma quístico con muerte fetal intrauterina y su seguimiento desde el punto de vista clínico y anatomopatológico

Monoterpene Glucosides from Cunila spicata.

The leaves of CUNILA SPICATA L. (Lamiaceae) yielded five monoterpene glucosides: three esters of oleuropeic acid with glucose ( 1, 2/3) as well as betulalbuside A ( 4) and roseoside A ( 5). Structure elucidation was done by high-field NMR-techniques.

Echinodol: A New Cembrene Derivative from Echinodorus grandiflorus.

The aerial parts of ECHINODORUS GRANDIFLORUS Mitch., a species of Alismataceae from Brazil, yielded a new diterpene alcohol named echiriodol ( 1) in addition to the known steroid stigmasterol. Its constitution and relative configuration were established by high resolution NMR techniques.

New Monoterpenes from Cunila spicata.

The leaves of CUNILA SPICATA Benth. (Lamiaceae) yielded a monoterpene diacetate called iso-rosiridol diacetate ( 1) and two isomeric hydroperoxymonoterpenes ( 2, 3) as well as beta-sitosterol, phytol, cadinol, linalool, dihydrocarveol, geranyl and neryl acetate. Structure elucidation was done by high-field NMR-techniques.

The Constitution and Configuration of Isocaryophyllen-13-al.

The aerial parts of CUNILA SPICATA Benth., a Brasilian Lamiaceae, afforded a sesquiterpene-aldehyde named isocaryophyllen-13-al (beta-betulenal) ( 1). The constitution and configuration of this compound were established by high field NMR-techniques for the first time.

[Peroxides as plant constituents. 8. Guaianolide-peroxides from yarrow,Achillea millefolium L., a soluble component causing yarrow dermatitis]. / Guaianolid-Peroxide aus der Schafgarbe, Achillea millefolium L., Auslöser der Schafgarbendermatitis.

From the ether extract of the blossoms of yarrow, Achillea millefolium L., two guaianolides (1, 2) with a peroxide bridged cyclopentane ring and an alpha-methylene-gamma-butyrolactone structure have been isolated. For these compounds the names alpha-peroxyachifolid (1) and beta-peroxyisoachifolid (2) are proposed. 1 is responsible for the allergic contact dermatitis caused by yarrow.