Spatial and temporal changes in cell proliferation in the chick jejunum during the folding of the ridges into zigzags.

Vilification in the chick gut involves the formation of longitudinal ridges, establishment of their zigzag pattern and emergence of individual villi. Although the morphological changes during vilification are well known in the chick gut, the pattern of cell proliferation during this process is poorly understood. The aim of the present study was to correlate spatial and temporal changes in cell proliferation to folding of the longitudinal ridges into zigzags. Embryos on the 13th pos-incubation day were injected with BrdU and sacrificed at 8 h intervals up to 64 h after injection. Spatial and temporal changes in cell proliferation were observed during the folding the longitudinal ridges into zigzags. Cell proliferation occurred throughout the epithelium of the folded ridges, was predominant in the epithelial cells at the sides of the zigzagging ridges, and finally appeared in the epithelial cells at the tips of the zigzag ridges. In conclusion, cell proliferation might be a requirement for the folding of the longitudinal ridges into zigzags.

Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.

Homeopathic Individualized Q-Potencies versus Fluoxetine for Moderate to Severe Depression: Double-Blind, Randomized Non-Inferiority Trial.

Homeopathy is a complementary and integrative medicine used in depression, The aim of this study is to investigate the non-inferiority and tolerability of individualized homeopathic medicines [Quinquagintamillesmial (Q-potencies)] in acute depression, using fluoxetine as active control. Ninety-one outpatients with moderate to severe depression were assigned to receive an individualized homeopathic medicine or fluoxetine 20 mg day(-1) (up to 40 mg day(-1)) in a prospective, randomized, double-blind double-dummy 8-week, single-center trial. Primary efficacy measure was the analysis of the mean change in the Montgomery & Åsberg Depression Rating Scale (MADRS) depression scores, using a non-inferiority test with margin of 1.45. Secondary efficacy outcomes were response and remission rates. Tolerability was assessed with the side effect rating scale of the Scandinavian Society of Psychopharmacology. Mean MADRS scores differences were not significant at the 4th (P = .654) and 8th weeks (P = .965) of treatment. Non-inferiority of homeopathy was indicated because the upper limit of the confidence interval (CI) for mean difference in MADRS change was less than the non-inferiority margin: mean differences (homeopathy-fluoxetine) were -3.04 (95% CI -6.95, 0.86) and -2.4 (95% CI -6.05, 0.77) at 4th and 8th week, respectively. There were no significant differences between the percentages of response or remission rates in both groups. Tolerability: there were no significant differences between the side effects rates, although a higher percentage of patients treated with fluoxetine reported troublesome side effects and there was a trend toward greater treatment interruption for adverse effects in the fluoxetine group. This study illustrates the feasibility of randomized controlled double-blind trials of homeopathy in depression and indicates the non-inferiority of individualized homeopathic Q-potencies as compared to fluoxetine in acute treatment of outpatients with moderate to severe depression.

Homeopathic Individualized Q-potencies versus Fluoxetine for Moderate to Severe Depression: Double-blind, Randomized Non-inferiority Trial

Homeopathy is a complementary and integrative medicine used in depression, The aim of this study is to investigate the non-inferiority and tolerability of individualized homeopathic medicines [Quinquagintamillesmial (Q-potencies)] in acute depression, using fluoxetine as active control. Ninety-one outpatients with moderate to severe depression were assigned to receive an individualized homeopathic medicine or fluoxetine 20 mg day(-1) (up to 40 mg day(-1)) in a prospective, randomized, double-blind double-dummy 8-week, single-center trial. Primary efficacy measure was the analysis of the mean change in the Montgomery & Asberg Depression Rating Scale (MADRS) depression scores, using a non-inferiority test with margin of 1.45. Secondary efficacy outcomes were response and remission rates [...] Non-inferiority of homeopathy was indicated because the upper limit of the confidence interval (CI) for mean difference in MADRS change was less than the non-inferiority margin: mean differences (homeopathy-fluoxetine) were -3.04 (95% CI -6.95, 0.86) and -2.4 (95% CI -6.05, 0.77) at 4th and 8th week, respectively. There were no significant differences between the percentages of response or remission rates in both groups. Tolerability: there were no significant differences between the side effects rates, although a higher percentage of patients treated with fluoxetine reported troublesome side effects and there was a trend toward greater treatment interruption for adverse effects in the fluoxetine group. This study illustrates the feasibility of randomized controlled double-blind trials of homeopathy in depression and indicates the non-inferiority of individualized homeopathic Q-potencies as compared to fluoxetine in acute treatment of outpatients with moderate to severe depression.(AU)

Treatment of suckling beef cattle with a progestagen sponge and oestradiol benzoate or equine chorionic gonadotrophin.

The ovarian responses of anoestrus beef cows to a combined treatment with medroxy-progesterone acetate (MAP) sponges and oestradiol benzoate or equine chorionic gonadotrophin (eCG) were evaluated. Forty-five suckling Hereford cows were allocated to three equal groups. Group 1 received a MAP sponge for seven days plus an injection of 2 mg oestradiol benzoate when the sponge was inserted (day 0) and 1 mg when the sponge was withdrawn; group 2 received identical treatment until day 7, when a dose of 400 iu of eCG was administered, and group 3 were left untreated as control animals. From day 0 to day 11 the cows' ovaries were examined daily by transrectal ultrasonography, and their oestrous behaviour was observed from 24 hours to 96 hours after the sponge was removed. Data from cows that had a corpus luteum present before the sponge was withdrawn were not used in subsequent analyses; there were four in group 1, five in group 2 and four in group 3. In 19 of the 21 cows in groups 1 and 2 a new follicular wave was observed to emerge at a mean (sd) interval of 3.9 (0.3) days after the insertion of the sponge, whereas in group 3 it occurred in all 11 cows after 3.4 (0.6) days. Only the six cows that had a follicle of 9 mm or larger in diameter ovulated (P < or = 0.001). Nine of the 11 cows in group 1 came into oestrus, compared with two of the 10 in group 2 and none of the control cows (P < or = 0.001). Ovulation was observed in four, two and none of the cows in groups 1, 2 and 3, respectively.

Use of nonergot alkaloid-producing endophytes for alleviating tall fescue toxicosis in stocker cattle.

Grazing studies were conducted to determine cattle growth performance, evaluate toxicosis, and compare grazing behavior in stocker cattle grazing nonergot alkaloid-producing endophyte-infected (AR542 or AR502), endophyte-free (E-), or wild-type toxic endophyte-infected (E+) Jesup, Georgia-5, and Kentucky-31 tall fescue. Replicated 0.81-ha tall fescue paddocks were established at the Central Georgia Branch Station at Eatonton and the Northwest Georgia Branch Station at Calhoun during October 1998 and were stocked with beef cattle for autumn and spring periods from fall 1999 through spring 2002. Mean ergot alkaloid concentrations were higher (P < 0.01) on E+ pastures than the other treatments at both locations. At Calhoun and Eatonton, post-treatment serum prolactin concentrations were decreased (P < 0.01) on E+ compared with AR542, AR502, and E- tall fescue. Cattle on AR542, AR502, and E- pastures had lower (P < 0.05) post-treatment rectal temperatures than cattle grazing E+ tall fescue during spring at Eatonton and Calhoun. Calf ADG was higher (P < 0.05) on AR542, AR502, and E- as compared with E+ tall fescue during autumn and spring grazing at Eatonton, and at Calhoun, cattle on E+ pastures had lower (P < 0.05) ADG in both autumn and spring. Gain/hectare was higher (P < 0.05) on AR542, AR502, and E- than on E+ during autumn at Eatonton and during spring at both locations. In autumn at Calhoun, gain/hectare was greater (P < 0.05) on AR502 and E- compared with E+ tall fescue. During April, May, and June, cattle grazing E+ pastures at Eatonton spent more (P < 0.01) time idling, more (P < 0.01) time standing, and used more (P < 0.01) water than cattle on AR542 and E- tall fescue. Daily prehensions and biting rate were each higher (P < 0.01) on AR542 and E- tall fescue than E+ tall fescue in both grazing seasons. There were no differences among pasture treatments for bite size in either spring (P = 0.50) or autumn (P = 0.34). Steers grazing E+ pastures had lower DMI than steers grazing AR542 and E- pastures during spring (P < 0.10) and lower DMI than steers grazing E- pastures during autumn (P < 0.05). Daily steer water usage was decreased (P < 0.10) in E+ pastures compared with AR542 and E- pastures during late fall. These results indicate that nonergot alkaloid-producing endophyte technology is a promising option for alleviating tall fescue toxicosis in stocker cattle.

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco.

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

An enantiomeric assay for the flavonoids medicarpin and vestitone using capillary electrophoresis.

An enantiomeric assay for the flavonoids vestitone and medicarpin from transgenic plant extracts was developed using capillary electrophoresis. It was found that no single cyclodextrin proved capable of resolving the enantiomers of both medicarpin and vestitone. Instead, hydroxypropyl-beta-cyclodextrin provided the best selectivity for the vestitones while hydroxypropyl-gamma-cyclodextrin was best for the medicarpins. The addition of organic modifiers improved the resolution of both enantiomers. Acetonitrile proved best for the vestitones and only methanol improved the resolution of the medicarpins. An optimization study of mixed hydroxypropyl-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin containing electrolytes revealed that the separation of the medicarpin enantiomers was intolerant to the presence of hydroxypropyl-beta-cyclodextrin. Our optimized running electrolyte was composed of 2 mM hydroxypropyl-beta-cyclodextrin, 20 mM hydroxypropyl-gamma-cyclodextrin, and 25 mM borate at pH 10.0 with 10% v/v methanol. This system provided a resolution of 1.47 and 1.80 for the medicarpin and vestitone enantiomers, respectively. This analysis was completed in 12 min. This separation provided a rapid screen to determine the enantiomeric purity of key flavonoids biosynthesized by transgenic legumes.

Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis.

Alfalfa (Medicago sativa) was transformed with a peanut (Arachis hypogaea) cDNA encoding resveratrol synthase (RS) transcriptionally regulated by an enhanced Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated a new compound, not present in wild-type or vector-transformed alfalfa, that was identified as trans-resveratrol-3-O-beta-D-glucopyranoside (RGluc) by high-pressure liquid chromatography (HPLC), UV, 1H- and 13C-nuclear magnetic resonance (NMR) analyses. RGluc concentration was highest in the youngest leaves (>15 microg per g fresh weight) and oldest stem internode segments (>10 microg per g fresh weight) while roots contained only trace amounts (<0.2 microg per g fresh weight). RS transcript levels were highest in leaves and stems, with comparatively little transcript accumulation in the roots, while an inverse pattern was observed for chalcone synthase (CHS) transcript levels. CHS directly competes with RS for the metabolic precursors p-coumaroyl CoA and malonyl CoA, and may also contribute to the developmental variations in RGluc levels by limiting the availability of substrates. Agar-plate bioassays indicated that both RGluc and resveratrol greatly inhibit hyphal growth of the alfalfa fungal pathogen Phoma medicaginis. Subsequently, RGluc-containing leaves were wound inoculated and showed a significant reduction (relative to control leaves) in the size of necrotic lesions, intensity of adjacent chlorosis, and number of fungal reproductive structures (pycnidia). Decreasing sporulation of this pathogen may greatly reduce disease spread and severity throughout the field.

Poisoning of cattle and sheep in Uruguay by sawfly (Perreyia flavipes) larvae.

From June to early October of 1993, 1994 and 1995 at least 40 outbreaks of a highly lethal disease occurred in cattle and sheep in the central region of Uruguay. During 1995 total cattle losses probably exceeded 1000 head. Mortalities were 1.6%, 7.0% and 1.3% for calves, yearlings and adults, respectively, but mortalities up to 28% occurred on some farms. Sheep were less frequently affected than cattle. Most animals were just found dead. Cattle had weakness, muscular tremors, depression, stupor and death. Others became highly excited and aggressive. Most affected cattle died within 2 d. Jaundice and mild photosensitization were observed in cattle that survived longer. Gross and microscopic lesions were severe periacinar or massive necrosis of hepatocytes with prominent edema of the gall bladder wall and its attachments. Edema, ecchymoses and petechiae on serous membranes, ascites and dry content of the omasum, colon and rectum were also observed. Invariably larval body fragments and heads of P flavipes were found in the rumen and omasum. The diagnosis of sawfly poisoning was confirmed by experimental feeding of 3 sheep and 2 calves with 9 to 40 g of P flavipes larvae/kg body weight.

Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses--a review.

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. the recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

High-performance liquid chromatography/continuous-flow liquid secondary ion mass spectrometry of flavonoid glycosides in leguminous plant extracts.

A method for the identification of flavonoid glycosides utilizing continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) is presented. Minimum detectable quantities (MDQs) were determined for three model flavonoid glycosides (rutin, naringin and esculin) by both positive ion direct insertion probe (DIP)-LSIMS (1.6 nmol, 1.7 nmol and 730 pmol, respectively) and positive ion CF-LSIMS (330 pmol, 340 pmol and 290 pmol, respectively). Optimization of CF-LSIMS instrumental parameters was performed using the model compound rutin. Parameters optimized included mobile phase composition, glycerol concentration, mobile phase flow rate, ion source temperature, acceleration lens potential (amplitude and polarity) and Cs+ primary ion energy. Final instrumental optimization yielded an MDQ of 1.0 ng (1.6 pmol) for rutin by flow-injection CF-LSIMS. The optimization parameters were utilized in the identification of flavonoid glucosides in alfalfa (Medicago sativa L) and chickpea (Cicer arietinum) extracts by high-performance liquid chromatogrphy/CF-LSIMS. The results support the controversial identification of a major extract component as formononetin-7-O-glucoside-6"-malonate as opposed to afrormosin-7-O-glucoside-6"-malonate.

Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis.

Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism. One of the final steps of medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol, is catalyzed by vestitone reductase. A 1245-bp cDNA clone which encodes vestitone reductase was identified utilizing internal amino acid sequence of purified vestitone reductase. When expressed in Escherichia coli, the cloned enzyme exhibits strict substrate stereospecificity for (3R)-vestitone, as was observed for vestitone reductase purified from alfalfa. The calculated molecular weight of the protein (35,918) is similar to that of purified vestitone reductase from alfalfa (38 kDa by SDS-PAGE). The levels of vestitone reductase transcript (1.35 kb) greatly increase within 2 h of elicitor addition to alfalfa cell suspension cultures, preceding the rapid increases in vestitione reductase enzyme activity and medicarpin biosynthesis. In healthy alfalfa plants, the highest levels of transcripts were detected in roots and root nodules, consistent with the synthesis of medicarpin and its conjugate in these tissues. The cloning of the vestitone reductase gene provides a specific tool for the study and manipulation of pterocarpan biosynthesis in legumes.

Vigilancia epidemiológica de las infecciones intrahospitalarias: estudios sistematizados de prevalencia (1993-1994) / Epidemiological surveillance in intrahospital infections: systemic studies of prevalence (1993-1994)

A partir de la sistematización de los estudios de prevalencia en 1993, continuamos con la presentación de los datos obtenidos en 1994, en conjunto con los del peíodo anterior. Esto permite tener una visualización gráfica de los resultados obtenidos pudiéndose analizar así sus tendencias. Si bien la metodología básica de los estudios no se ha modificado, a partir de diciembre de 1993 se incorporó al análisis, un sistema subjetivo que permite estratificar el nivel de gravedad de los pacientes y relacionarlo con el riesgo de infección intrahospitalaria. Este modelo fue validado a partir de los estudios de diciembre de 1993, abril y agosto de 1994, evaluándose además su reproducibilidad. El análisis comparativo de los estudios de prevalencia, muestra la persistencia de una proporción elevada de pacientes con acceso vascular (>50 por ciento) y catéter urinario (>20 por ciento), lo que determina un riesgo incrementado de desarrollar infecciones asociadas a estos factores. La tasa de prevalencia de infecciones intrahospitalarias se mantuvo por encima del 10 por ciento...

Vigilancia epidemiológica de las infecciones intrahospitalarias: estudios sistematizados de prevalencia (1993-1994) / Epidemiological surveillance in intrahospital infections: systemic studies of prevalence (1993-1994)

A partir de la sistematización de los estudios de prevalencia en 1993, continuamos con la presentación de los datos obtenidos en 1994, en conjunto con los del peíodo anterior. Esto permite tener una visualización gráfica de los resultados obtenidos pudiéndose analizar así sus tendencias. Si bien la metodología básica de los estudios no se ha modificado, a partir de diciembre de 1993 se incorporó al análisis, un sistema subjetivo que permite estratificar el nivel de gravedad de los pacientes y relacionarlo con el riesgo de infección intrahospitalaria. Este modelo fue validado a partir de los estudios de diciembre de 1993, abril y agosto de 1994, evaluándose además su reproducibilidad. El análisis comparativo de los estudios de prevalencia, muestra la persistencia de una proporción elevada de pacientes con acceso vascular (>50 por ciento) y catéter urinario (>20 por ciento), lo que determina un riesgo incrementado de desarrollar infecciones asociadas a estos factores. La tasa de prevalencia de infecciones intrahospitalarias se mantuvo por encima del 10 por ciento...(AU)

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.