The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism

cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.     

The<Emphasis Type="Italic"> Rxo1</Emphasis>/<Emphasis Type="Italic">Rba1</Emphasis> locus of maize controls resistance reactions to pathogenic and non-host bacteria

The genetic mechanism underlying six palatability properties of cooked rice and three physico-chemical traits was dissected in 66 BC₃F₂ chromosome segment substitution lines (CSSLs), using a complete linkage map in three successive years. The CSSLs showed transgressive segregation for all traits studied. Significant correlation was detected among most palatability traits. A total of 25 QTLs for the nine traits were identified on nine chromosomes, and many QTLs affecting different quality traits were mapped in the same regions. Six QTLs—qLT-8 for luster, qTD-6 and qTD-8 for tenderness, qIVOE-6 and qIVOE-8 for integrated value of organoleptic evaluation, and qAC-8 for amylose content—were repeatedly detected across the 3 years. Phenotypic values were significantly different between the recurrent parent, cultivar Asominori, and the CSSLs harboring any of the six QTL alleles across the three environments, indicating that these six QTLs were non-environment-specific and could be used for marker-assisted selection in rice quality improvement.     

Histamine enhances cytotrophoblast invasion by inducing intracellular calcium transients through the histamine type-1 receptor

Blastocyst implantation and placentation require molecular and cellular interactions between the uterine endometrium and blastocyst trophectoderm. Previous studies showed that histamine produced in the mouse uterine luminal epithelium interacts with trophoblast histamine type-2 receptors (H2) to initiate blastocyst implantation. However, it is unknown whether similar histamine activity is operative in humans. Using a human cell line (HTR-8/SVneo) derived from first-trimester cytotrophoblasts that expresses both histamine type-1 receptor (H1) and H2, we found that histamine promotes cytotrophoblast invasiveness specifically through activation of H1. Stimulation of H1 in human cytotrophoblasts by histamine induced intracellular Ca2+ (Ca(2+)i) transients by activating phospholipase C and the inositol trisphosphate pathway. The enhanced invasion induced by histamine was blocked by pretreatment with H1 antagonist or by chelation of Ca(2+)i. These findings suggest possible differences between rodents and humans in histamine signaling to the trophoblast.     

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.     

Vibrating membrane filtration for recovery and concentration of insect killing nematodes

New experimental data are reported that demonstrate the use of a novel vibrating membrane filter (VMF) for the combined recovery and concentration of two species of nematodes, S. feltiae and P. hermaphrodita, from mature liquid fermentation cultures. The disk membrane module had a working surface area of 0.2 m(2) and was operated at a constant flow rate of 0.2 m(3) h(-1). The recovery of the viable nematodes from the spent media and nonviable nematodes was assisted by an independently imposed oscillatory motion of the disk assembly, which produced an intense shear field at the membrane surface with calculated mean values on the order of 10(4) s(-1). Adult (nonviable) nematodes in the fermentation culture were preferentially dissolved in a detergent (sodium dodecylsulfate) and successfully separated from the juveniles using the VMF equipment. Permeate fluxes on the order of 15 to 30 L/m(2/)h were achieved for an operating transmembrane pressure of 800 mbar. Industrial-scale liquid fermentation for the manufacture of nematodes as biopesticides produces the viable nematode life stages in low-concentration suspension containing large quantities of spent media and other waste material. The VMF equipment provided a flexible operation for separation, cleaning, and concentration of viable nematodes from the fermentation broths.     

Bioinformatic analyses of the tRNA: (guanine 26, N2,N2)-dimethyltransferase (Trm1) family

Functional analyses of the tRNA:(guanine 26, N2,N2)-dimethyltransferase (Trm1) have been hampered by a lack of structural information about the enzyme and by low sequence similarity to better studied methyltransferases. Here we used computational methods to detect novel homologs of Trm1, infer the evolutionary relationships of the family, and predict the structure of the Trm1 methyltransferase. The N-terminal region of the protein is predicted to form an S-adenosylmethionine-binding domain, which harbors the active site. The C-terminal region is rich in predicted alpha-helices and, in analogy to other nucleic acid methyltransferases, may constitute the target recognition domain of the enzyme. Interposing these two domains, most Trm1 homologs possess a highly variable inserted sequence that is delimited by a Cys4 cluster, likely forming a Zn-finger structure. The residues of Trm1 predicted to participate in cofactor binding, target recognition, and catalysis, were mapped onto a preliminary structural model, providing a platform for designing new experiments to better understand the molecular functions of this protein family. In addition, identification of novel, atypical Trm1 homologs suggests candidates for cloning and biochemical characterization.     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.     

Reactions of e(-)(aq), CO(2)(*)(-), HO(*), O(2)(*)(-) and O(2)((1)delta(g)) with a dendro[60]fullerene and C(60)[C(COOH)(2)](n) (n = 2-6)

Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process.     

Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness

Genomic copies of three Xanthomonas oryzae pv. oryzae avirulence (avr) genes, avrXa7, avrXal0, and avrxa5, and four homologous genes, aB3.5, aB3.6, aB4.3, and aB4.5, were mutagenized individually or in combination to study the roles of avr genes in one component of pathogen fitness, i.e., aggressiveness or the amount of disease X. oryzae pv. oryzae causes in susceptible rice lines. These X. oryzae pv. oryzae genes are members of the highly related Xanthomonas avrBs3 gene family. Compared to the wild-type strain, X. oryzae pv. oryzae strains with mutations in avrXa7, avrxa5, and the four homologous genes caused shorter lesions on rice line IR24, which contains no resistance genes relevant to the wild-type strain. The contribution of each gene to lesion length varied, with avrXa7 contributing the most and avrXal0 showing no measurable effect on aggressiveness. The functional, plasmidborne copies of avrXa7, aB4.5, and avrxa5 restored aggressiveness only to strains with mutations in avrXa7, aB4.5, and avrxa5, respectively. Mutations in avrXa7 were not complemented by plasmids carrying any other avr gene family members. These data indicate that some, but not all, avr family members contribute to pathogen aggressiveness and that the contributions are quantitatively different. Furthermore, despite their sequence similarity, the aggressiveness functions of these gene family members are not interchangeable. The results suggest that selection and pyramiding resistance genes can be guided by the degree of fitness penalty that is empirically determined in avr gene mutations.     

Localized reversible frameshift mutation in the flhA gene confers phase variability to flagellin gene expression in Campylobacter coli

Phase variation of flagellin gene expression in Campylobacter coli UA585 was correlated with high-frequency, reversible insertion and deletion frameshift mutations in a short homopolymeric tract of thymine residues located in the N-terminal coding region of the flhA gene. Mutation-based phase variation in flhA may generate functional diversity in the host and environment.