Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys¹⁶⁵, a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys¹⁶⁵ modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys¹⁶⁵ was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys¹⁶⁵ is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys¹⁶⁵. The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.     

Detection of anti-leishmanial effect of the Lucilia sericata larval secretions in vitro and in vivo on Leishmania tropica: First work

It is known that some of the enzymes and substances secreted by 2nd and 3rd stages of the Lucilia sericata larvae to have bacteriostatic and bactericidal effects. From this point of view, we investigated the anti-leishmanial effect of larval secretions of the L. sericata on the Leishmania tropica both in vitro and in vivo conditions. In vitro: It was observed that promastigotes of L. tropica had undergone lyzis within 1min in the larval secretions of L. sericata. However, larval secretion was ineffective on the promastigotes within Novy-MacNeal-Nicolle (NNN) cultures and RPMI 1640 medium. In vivo: Seven groups of male Balb/C mice (6 study groups and 1 control group), each composed of eight weeks old 10 mice were formed. L. tropica promastigotes were injected subcutaneosly to the soles of the SG mice' feet. In study groups, cutaneous lesions were developed Limoncu et al., 1997 in 2 (20%) and 1 (10%) of the SG-1 and SG-2, respectively after 15days. There were L. tropica in the smears prepared from the lesions and L. tropica was observed in the cultures. Cutaneous lesions were not developed in 8 (80%), 9 (90%) and 10 (100%) of the SG-I, SG-II and SG-III, respectively. There were no cutaneous lesions developed in the soles of the feet. There were no L. tropica in the smears prepared from the infected soles of the feet neither L. tropica was observed in the cultures. Larval secretions were given into the cutaneous lesions to the feet soles of the SG-IV, V and VI mice after 6months. No healing was observed in the cutaneos lesions of 4 (40%), 5 (50%) and 1 (10%) of SG-IV, SG-V and SG-VI, after 6months, respectively. There were L. tropica in the smears prepared from the lesions and L. tropica was observed in the cultures. On the other hand, the lesions of 6 (60%), 5 (50%) and 9 (90%) of SG-IV, SG-V and SG-VI were diminished in size and disappeared completely after 6months. There were L. tropica observed in the smears prepared from the infected soles of the feet and no growth was observed in the cultures. In the smears prepared from the cutaneous lesions developed in the soles of the feet of the control group mice, L. tropica was visualized and observed in the cultures. A statistical significant difference was observed between study groups and control group (p<0.001). In our study we demonstrated for the first time that the secretions of the 2nd and 3rd stages sterile and pure larvae of L. sericata had effects on promastigotes of L. tropica in in vitro and very effective on amastigote forms in in vivo conditions.     

Mathematical modeling of the indole-3-butyric acid applications on rooting of northern highbush blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.) softwood-cuttings

The objective of the present study is to develop a mathematical model to predict the effect of indole-3-butyric acid (IBA) on mean rooting (%) and mean root growth of northern highbush blueberry cultivars (Vaccinium corymbosum L.). The best estimating equations for the rooting (%) and root growth are formulized as: RG = (5.672183) + [0.002851 × (IBA)] − [2.0E⁻⁶ × (IBA)2] + (−0.27211 × Cv.) and R = (82.00649) + [0.030801 × (IBA)] − [2,4E⁻⁵ × (IBA)2] − [2.36218 × (Cv.)] where RG is root growth, R is rooting, IBA is indole-3-butyric acid (ppm) and Cv. is cultivar. Cultivars are Ivanhoe [1], Jersey [2], Rekord [3], Northland [4], Berkeley [5] and Bluejay [6]. The numbers given in square brackets represent the blueberry cultivars for the equations. Multiple regression analysis was carried out until the least sum of squares (R2) was obtained. R ² value 0.90 for rooting and 0.95 for root growth. Standard errors were found to be significant at the p < 0.001 level. The actual rooting differed to the blueberry cultivars and it was between 57.76 and 83.23% while estimated rooting percentage calculated by the produced mathematical model was between 59.04 and 83.80%.     

Survey of insect growth regulator (IGR) resistance in house flies (Musca domestica L.) from southwestern Turkey

Insect growth regulators (IGRs) are currently the fastest‐growing class of insecticides, and in Turkey these products represent a new approach to pest control. In recent years, several IGRs were also registered for the control of the house fly, Musca domestica L. (Diptera: Muscidae), in Turkey. A field survey was conducted in the summers of 2006 and 2007 to evaluate resistance to some agriculturally and medically used IGRs on house flies from livestock farms and garbage dumps in the greenhouse production areas (Merkez, Kumluca, Manavgat, and Serik) of Antalya province (Southwestern Turkey). The results of larval feeding assay with technical diflubenzuron, methoprene, novaluron, pyripoxyfen, and triflumuron indicate that low levels (RF<10‐fold) of resistance to the IGRs exist in the house fly populations from Antalya province. Exceptions, however, were two populations, Guzoren and Toptas, from the Kumluca area which showed moderate resistance to diflubenzuron with 11.8‐fold in 2006 and 13.2‐fold in 2007, respectively. We found substantial variation in susceptibility of field‐collected house fly populations from year to year and from product to product. We generally observed an increase in resistance at many localities sampled from 2006 to 2007. The implications of these results to the future use of IGRs for house fly control are discussed. It will be critically important to continue monitoring efforts so that appropriate steps can be taken if resistance levels start to increase.     

Construction of Genetic Linkage Maps and Comparative Genome Analysis of Catfish Using Gene-Associated Markers

A genetic linkage map of the channel catfish genome (N = 29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3–18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.     

<Emphasis Type="Italic">MEFV</Emphasis> mutations in patients with familial Mediterranean fever from the Aegean region of Turkey

Familial Mediterranean Fever (FMF) which is frequently present in Mediterranean populations is caused by mutations in the MEFV gene. According to recent data, MEFV mutations are not the only cause of FMF, but these are major genetic determinants which cause FMF. It has also been suggested that there may be a number of other genes causing FMF. The MEFV gene is located at 16p13.3 and encodes a protein, pyrin/marenostrin. More than 70 disease associated mutations and totally 186 mutations and polymorphisms have been defined in affected individuals. We have retrospectively evaluated the molecular test results of 1,201 patients identified as having FMF clinical symptoms referred to the Molecular Genetics Laboratory of the Department of Medical Genetics, Faculty of Medicine, Ege University, Izmir/Turkey over the last 4 years. Patients were tested for 12 common mutations in the MEFV gene using a strip assay method (Innogenetics, Belgium). Out of the 1,201 patients tested (2,402 chromosomes) in the Aegean region in Turkey, 654 (54.45%) did not carry any mutations, among the 547 (45.55%) patients with mutations 246 patients were either homozygous (101) or compound heterozygous (145), 296 carried only one detected mutation, and five patients had three mutations. Allelic frequencies for the four most common mutations in the mutation positive groups were 47.60% (M694V), 16.75% (E148Q), 12.95% (V726A), 11.94% (M680I G/C).The remaining alleles (10.76%) showed rare mutations which were R761H, P369S, A744S, K695R, F479L, M694I. When the frequencies of mutations detected in our group were compared to the frequencies reported in the other regions of Turkey, an increase in V726A mutation frequency was observed. No patient showed a I692del mutation which is sometimes evident in other Mediterranean populations.