Prothoracicotropic Hormone Acts as a Neuroendocrine Switch between Pupal Diapause and Adult Development

Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage.     

On the origin of DNA genomes: evolution of the division of labor between template and catalyst in model replicator systems

The division of labor between template and catalyst is a fundamental property of all living systems: DNA stores genetic information whereas proteins function as catalysts. The RNA world hypothesis, however, posits that, at the earlier stages of evolution, RNA acted as both template and catalyst. Why would such division of labor evolve in the RNA world? We investigated the evolution of DNA-like molecules, i.e. molecules that can function only as template, in minimal computational models of RNA replicator systems. In the models, RNA can function as both template-directed polymerase and template, whereas DNA can function only as template. Two classes of models were explored. In the surface models, replicators are attached to surfaces with finite diffusion. In the compartment models, replicators are compartmentalized by vesicle-like boundaries. Both models displayed the evolution of DNA and the ensuing division of labor between templates and catalysts. In the surface model, DNA provides the advantage of greater resistance against parasitic templates. However, this advantage is at least partially offset by the disadvantage of slower multiplication due to the increased complexity of the replication cycle. In the compartment model, DNA can significantly delay the intra-compartment evolution of RNA towards catalytic deterioration. These results are explained in terms of the trade-off between template and catalyst that is inherent in RNA-only replication cycles: DNA releases RNA from this trade-off by making it unnecessary for RNA to serve as template and so rendering the system more resistant against evolving parasitism. Our analysis of these simple models suggests that the lack of catalytic activity in DNA by itself can generate a sufficient selective advantage for RNA replicator systems to produce DNA. Given the widespread notion that DNA evolved owing to its superior chemical properties as a template, this study offers a novel insight into the evolutionary origin of DNA.     

Thermodynamics of mucopolysaccharide–dye binding. III. Thermodynamic and cooperativity parameters of acridine orange–heparin system

Binding isotherms for acridine orange (AO)–heparin systems can be evaluated solely on the basis of quantitative fluorescence spectroscopic measurements. The evaluation of thermodynamic parameters indicates that the interactions of AO with heparins from several animal sources are similar to each other in magnitude. Binding is highly exothermic (ΔH = ?6 kcal mol^?1) and is stabilized by dye–polymer and dye–dye (coopertive) interactions, as well as by entropic factors (ΔS = +7 e.u.). The predominant stabilizing factor appears to be the electrostatic attraction between the AO cation and the heparin polyanion, although the other factors are important as well. At 24°C the value of the cooperative binding constants for the various heparins range from 8.8 to 11.3 × 10⁵M^?1, corresponding to a free energy of ?8 kcal mol^?1. The degree of cooperativity, which is a direct measure of dye–dye interaction, varies with polymer:dye ratio; the theoretical basis for this variation remains to be elucidated. Electrophoretic data indicate that each heparin sample consists of a mixture of species, each with its own charge density. This precludes definitive interpretation of observed small differences in the values of the thermodynamic parameters among the various samples until each sample can be resolved into its components.     

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants

Summary The recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al. 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant. We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment. Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment. This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported. In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB. When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity. This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene. We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.Abbreviations TLD thymineless death - UV ultraviolet light - Ap ampicillin - Km kanamycin - Sm streptomycin - Tc tetracycline - ^r resistant - ^s sensitive - kb kilobase - kdal kilodalton     

Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S

The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A→G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed. Received: 5 September 1996     

Chelating Agent Shock of Bacteriophage T5

When two strains of phage T5 (heat-susceptible form T5st(+) and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed "chelating agent shock" (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st(+) was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.     

Nature and intensity of selection pressure on CRISPR-associated genes

The recently discovered CRISPR-Cas adaptive immune system is present in almost all archaea and many bacteria. It consists of cassettes of CRISPR repeats that incorporate spacers homologous to fragments of viral or plasmid genomes that are employed as guide RNAs in the immune response, along with numerous CRISPR-associated (cas) genes that encode proteins possessing diverse, only partially characterized activities required for the action of the system. Here, we investigate the evolution of the cas genes and show that they evolve under purifying selection that is typically much weaker than the median strength of purifying selection affecting genes in the respective genomes. The exceptions are the cas1 and cas2 genes that typically evolve at levels of purifying selection close to the genomic median. Thus, although these genes are implicated in the acquisition of spacers from alien genomes, they do not appear to be directly involved in an arms race between bacterial and archaeal hosts and infectious agents. These genes might possess functions distinct from and additional to their role in the CRISPR-Cas-mediated immune response. Taken together with evidence of the frequent horizontal transfer of cas genes reported previously and with the wide-spread microscale recombination within these genes detected in this work, these findings reveal the highly dynamic evolution of cas genes. This conclusion is in line with the involvement of CRISPR-Cas in antiviral immunity that is likely to entail a coevolutionary arms race with rapidly evolving viruses. However, we failed to detect evidence of strong positive selection in any of the cas genes.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.