Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.     

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

 Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10⁻² s⁻¹. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting. Received: 13 July 2000 / Accepted: 12 October 2000     

Costs of Illness Due to Cholera, Costs of Immunization and Cost-Effectiveness of an Oral Cholera Mass Vaccination Campaign in Zanzibar

Background

The World Health Organization (WHO) recommends oral cholera vaccines (OCVs) as a supplementary tool to conventional prevention of cholera. Dukoral, a killed whole-cell two-dose OCV, was used in a mass vaccination campaign in 2009 in Zanzibar. Public and private costs of illness (COI) due to endemic cholera and costs of the mass vaccination campaign were estimated to assess the cost-effectiveness of OCV for this particular campaign from both the health care provider and the societal perspective.

Methodology/Principal Findings

Public and private COI were obtained from interviews with local experts, with patients from three outbreaks and from reports and record review. Cost data for the vaccination campaign were collected based on actual expenditure and planned budget data. A static cohort of 50,000 individuals was examined, including herd protection. Primary outcome measures were incremental cost-effectiveness ratios (ICER) per death, per case and per disability-adjusted life-year (DALY) averted. One-way sensitivity and threshold analyses were conducted. The ICER was evaluated with regard to WHO criteria for cost-effectiveness. Base-case ICERs were USD 750,000 per death averted, USD 6,000 per case averted and USD 30,000 per DALY averted, without differences between the health care provider and the societal perspective. Threshold analyses using Shanchol and assuming high incidence and case-fatality rate indicated that the purchase price per course would have to be as low as USD 1.2 to render the mass vaccination campaign cost-effective from a health care provider perspective (societal perspective: USD 1.3).

Conclusions/Significance

Based on empirical and site-specific cost and effectiveness data from Zanzibar, the 2009 mass vaccination campaign was cost-ineffective mainly due to the relatively high OCV purchase price and a relatively low incidence. However, mass vaccination campaigns in Zanzibar to control endemic cholera may meet criteria for cost-effectiveness under certain circumstances, especially in high-incidence areas and at OCV prices below USD 1.3.     

Mutations in cell division proteins FtsZ and FtsA inhibit φX174 protein-E-mediated lysis of Escherichia coli

Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites. These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites. Therefore, E. coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis. The ftsZ84 and the ftsA12 cell division mutant strains of E. coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division. The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division – rather than specific functions of cell division – plays an essential role in protein-E-mediated lysis. SulA-overproducing cells had a lysis-positive phenotype, the ring structure – but not the GTPase function - of FtsZ was impaired. Received: 14 April 1998 / Accepted: 9 June 1998     

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.     

Smog nitrogen and the rapid acidification of forest soil, San Bernardino Mountains, southern California

We report the rapid acidification of forest soils in the San Bernardino Mountains of southern California. After 30 years, soil to a depth of 25 cm has decreased from a pH (measured in 0.01 M CaCl2) of 4.8 to 3.1. At the 50-cm depth, it has changed from a pH of 4.8 to 4.2. We attribute this rapid change in soil reactivity to very high rates of anthropogenic atmospheric nitrogen (N) added to the soil surface (72 kg ha(-1) year(-1)) from wet, dry, and fog deposition under a Mediterranean climate. Our research suggests that a soil textural discontinuity, related to a buried ancient landsurface, contributes to this rapid acidification by controlling the spatial and temporal movement of precipitation into the landsurface. As a result, the depth to which dissolved anthropogenic N as nitrate (NO3) is leached early in the winter wet season is limited to within the top approximately 130 cm of soil where it accumulates and increases soil acidity.     

Inter-population variability of leaf morpho-anatomical and terpenoid patterns of Pistacia atlantica Desf. ssp. atlantica growing along an aridity gradient in Algeria

Three Algerian populations of female Pistacia atlantica shrubs were investigated in order to check whether their terpenoid contents and morpho-anatomical parameters may characterize the infraspecific variability. The populations were sampled along a gradient of increasing aridity from the Atlas mountains into the northwestern Central Sahara.As evidenced by Scanning Electron Microscopy, tufted hairs could be found only on seedling leaves from the low aridity site as a population-specific trait preserved also in culture. Under common garden cultivation seedlings of the high aridity site showed a three times higher density of glandular trichomes compared to the low aridity site. Increased aridity resulted also in reduction of leaf sizes while their thickness increased. Palisade parenchyma thickness also increases with aridity, being the best variable that discriminates the three populations of P. atlantica.Analysis of terpenoids from the leaves carried out by GC-MS reveals the presence of 65 compounds. The major compounds identified were spathulenol (23 μg g⁻¹ dw), α-pinene (10 μg g⁻¹ dw), verbenone (7 μg g⁻¹ dw) and β-pinene (6 μg g⁻¹ dw) in leaves from the low aridity site; spathulenol (73 μg g⁻¹ dw), α-pinene (25 μg g⁻¹ dw), β-pinene (18 μg g⁻¹ dw) and γ-amorphene (16 μg g⁻¹ dw) in those from medium aridity and spathulenol (114 μg g⁻¹ dw), α-pinene (49 μg g⁻¹ dw), germacrene D (29 μg g⁻¹ dw) and camphene (23 μg g⁻¹ dw) in leaves from the high aridity site. Terpene concentrations increased with the degree of aridity: the highest mean concentration of monoterpenes (136 μg g⁻¹ dw), sesquiterpenes (290 μg g⁻¹ dw) and total terpenes (427 μg g⁻¹ dw) were observed in the highest arid site and are, respectively, 3-, 5- and 4-fold higher compared to the lower arid site. Spathulenol and α-pinene can be taken as chemical markers of aridity. Drought discriminating compounds in low, but detectable concentrations are δ-cadinene and β-copaene. The functional roles of the terpenoids found in P. atlantica leaves and principles of their biosynthesis are discussed with emphasis on the mechanisms of plant resistance to drought conditions.