Open software for biologists: from famine to feast

Developing and deploying specialized computing systems for specific research communities is achievable, cost effective and has wide-ranging benefits.     

Three-Tube Method for Screening Stool Cultures for Salmonella and Shigella

A three-tube method developed is described as a screening test for non-lactose-fermenting organisms isolated from stool cultures. To evaluate the method, 976 strains of gram-negative bacilli were tested. All strains of Salmonella and Shigella were correctly identified generically.     

Preparation,characterization, and antitumor activity of new cisplatin analogues with 1-methyl-4-(methylamino)piperidine: crystal structure of [PtII(1-methyl-4-(methylamino) piperidine)(oxalate)

A series of new platinum(II) complexes of the type [Pt(II)(mmap)X] (where mmap, 1-methyl-4-(methylamino)piperidine and X, 1,1-cyclobutanedicarboxylato (CBDCA), oxalato, malonato, methylmalonato, dimethylmalonato, ethylmalonato, diethylmalonato or 2,3-naphthalene dicarboxylato (NDCA)) have been synthesized and characterized by elemental analysis, infrared (IR), and 13C and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the analogue [Pt(II)(mmap)(oxalate)] was determined using the single crystal X-ray diffraction method. Based upon a total of 4964 collected reflections, we determined that the compound crystallizes in the monoclinic space group P2(1)/c (with a=11.890(2) A, b=9.6695(19) A, c=9.875(2) A, beta=102.03(3) degrees, Z=4, and R=0.0428). In this complex, platinum has a slightly distorted square planar geometry with the two adjacent corners being occupied by two nitrogen atoms of the mmap ligand, whereas the remaining cis positions are occupied by two oxygen atoms of the oxalate molecule. The mmap ligand is in a boat conformation and forms six-membered chelating rings as well as the oxalate molecule forms five-membered chelating rings with platinum. The complexes were evaluated for their cytotoxic potential against the sensitive A2780 tumor model and cisplatin-resistant clone derived in vitro from potential cells.     

Sequence-selective recognition of duplex DNA through covalent interstrand cross-linking: kinetic and molecular modeling studies with pyrrolobenzodiazepine dimers

Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH(2))(n)-O-C8' diether linkages (n = 3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n = 3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n = 5) can span an extra base pair and cross-link the 5'-Pu-GA(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n = 3 (2a) > n = 5 (2c) > n = 6 (2d) > n = 4 (2b). The n = 3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n = 3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h(-1) microM(-1) at 37 degrees C, respectively), whereas the n = 4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h(-1) microM(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to configurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.     

Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells

In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI searches within the GenBank™ database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.     

A reexamination of the epithelial sensory cells of amphioxus (Branchiostoma)

The rostral epithelium of a newly metamorphosed juvenile of Branchiostoma floridae was examined at the EM level to confirm previous reports on its sensory cells. The majority of the sensory cells are of three types: two type I variants, with simple collars of unbranched microvilli surrounding their cilia, and one kind of type II cell, with an extended collar of repeatedly branched microvilli. The two type I variants differ in the structure and arrangement of the microvilli, basal body and rootlet, and the length of the cilium. Both variants are probably primary sensory cells (i.e. each has its own axon), but the data supporting this conclusion are much better for one variant than for the other. Type II cells are secondary sensory cells, with synaptic terminals borne on short extensions of the cell body. The presence of degenerating type II cells suggests that they may be subject to a regular process of loss and renewal. The results do not resolve the evolutionary issue of how amphioxus sensory cells relate to the epithelial sensory and receptor cells of vertebrates. Being primary, the type I cells resemble the supposed ancestral type more closely than do type II cells. Type II cells may be chemosensory, however, and should not be ruled out a priori as possible homologues of either primary or secondary chemosensory cells in vertebrates.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.     

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Doudoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with ¹⁴C in carbon atoms 1, 2, 3, 3+4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Doudoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - EDTA ethylene diamine tetra-acetic acid, disodium salt - FDP fructose 1,6-diphosphate - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - Pa Pascal (10⁵ Pa=1 bar) - PP pentose phosphate - POPOP 1,4-di[2-(5-phenyloxazolyl)] benzene - PPO 2,5-diphenyloxazole