Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.     

Leishmania donovani amastigotes mobilize organic and inorganic osmolytes during regulatory volume decrease

The protozoan parasite Leishmania donovani encounters large fluctuations in osmolality as it cycles between its insect vector and human host. The flagellated promastigote exhibits regulatory volume responses involving organic and inorganic osmolytes, but little is known about volume regulation in the clinically relevant amastigote that multiplies within the parasitophorous vacuoles of mammalian host cells. Using a combination of morphological, X-ray microanalytical, and biochemical approaches we determined that non-motile amastigotes respond to hypotonic stress with (1) an amino acid and l-alanine-mediated regulatory volume decrease, and (2) a parallel release of Na+, K+, P (presumably as negatively charged phosphates), and subsequently Cl- from cytoplasm and the cell as a whole. In addition P, Zn2+, and subsequently Ca2+ increase in acidocalcisomes as Cl- content declines in this compartment. This evidence is the first to document subcellular translocation of, and thus a potential role for, zinc in volume regulatory responses. These coordinated changes in organic and inorganic osmolytes demonstrate that amastigote subcellular compartments, particularly acidocalcisomes, function in maintaining ionic homeostasis in the response of Leishmania amastigotes to hypo-osmotic stress.     

An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid     

THE DEVELOPMENT OF APOSPOROUS GAMETOPHYTES AND REGENERATED SPOROPHYTES FROM EPIDERMAL CELLS OF EXCISED FERN LEAVES: AN ANATOMICAL STUDY

Three different types of outgrowths develop from epidermal cells of excised juvenile leaves of Microgramma vacciniifolia: aposporous gametophytes, intermediates, and regenerated sporophytic plantlets. The gametophytes and intermediates arise from derivatives of epidermal cell divisions which are developed to the exterior of the leaf surface, whereas the sporophytic regenerants originate from derivatives produced by cell divisions to the interior of the leaf. Anatomical observations of excised leaves grown in vitro demonstrate that only the epidermal cells are stimulated to divide and give rise to the various types of outgrowths. Incorporation of tritiated thymidine by the nuclei of leaf epidermal cells gives further evidence for the metabolic activity of these cells.     

Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.     

Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery

Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.     

Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.     

The 3-Phosphorylated Phosphoinositide Response of 3T3-L1 Preadipose Cells Exposed to Insulin,Insulin-Like Growth Factor-1, or Platelet-Derived Growth Factor

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P₂ either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P₃ peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P₃ response. Insulin failed to increase PI(3,4)P₂ at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P₂ and PI(3,4,5)P₃ rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P₃ with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P₂.