Escherichia coli phosphoglucose isomerase can be substituted by members of the PGI family, the PGI/PMI family, and the cPGI family

The Escherichia coli strain Pgi-UdhA, a mutant of the strain MG1655, is deficient in both the pgi gene and the udhA gene and cannot grow on glucose as carbon and energy source. This strain was transformed with different pET-plasmids containing archaeal or bacterial pgi, cpgi or pgi/pmi genes from the three known PGI families (PGI, PGI/PMI, cPGI). Growth could be restored upon plasmid-based expression of pgi, pgi/pmi or cpgi genes indicating that these heterologous proteins can substitute for E. coli PGI. However, complete restoration of the growth rate could not be obtained by any of the PGIs, PGI/PMIs, or cPGIs used. The data indicate that the PGI function of the three PGI families is functionally exchangeable in glycolysis.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

From the bottom of the heart: anteroposterior decisions in cardiac muscle differentiation

Recently, studies on specification of axes in the developing embryo have focused on the heart, which is the first functional organ to form and probably responds to common cues controlling positional information in surrounding tissues. The early differentiation of heart cells affords an opportunity to link the acquisition of regional identity with the signals underlying terminal differentiation. In the past year, a wealth of information on these signals has emerged, elucidating the general pathways controlling body axes in the context of the developing heart.     

Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. There is an increased risk of benign and malignant tumors in the gastrointestinal tract and in extraintestinal tissues. One PJS locus has been mapped to chromosome 19p13.3; a second locus is suspected on chromosome 19q13.4 in a minority of families. The PJS gene on 19p13.3 has recently been cloned, and it encodes the serine/threonine kinase LKB1. The gene, which is ubiquitously expressed, is composed of 10 exons spanning 23 kb. Several LKB1 mutations have been reported in heterozygosity in PJS patients. In this study, we screened for LKB1 mutations in nine PJS families of American, Spanish, Portuguese, French, Turkish, and Indian origin and detected seven novel mutations. These included two frameshift mutations, one four-amino-acid deletion, two amino-acid substitutions, and two splicing errors. Expression of mutant LKB1 proteins (K78I, D176N, W308C, and L67P) and assessment of their autophosphorylation activity revealed a loss of the kinase activity in all of these mutants. These results provide direct evidence that the elimination of the kinase activity of LKB1 is probably responsible for the development of the PJS phenotypes. In two Indian families, we failed to detect any LKB1 mutation; in one of these families, we previously had detected linkage to markers on 19q13.3-4, which suggests locus heterogeneity of PJS. The elucidation of the molecular etiology of PJS and the positional cloning of the second potential PJS gene will further elucidate the involvement of kinases/phosphatases in the development of cancer-predisposing syndromes.     

Cell polarity, intercellular signalling and morphogenetic cell movements in Myxococcus xanthus

In Myxococcus xanthus morphogenetic cell movements constitute the basis for the formation of spreading vegetative colonies and fruiting bodies in starving cells. M. xanthus cells move by gliding and gliding motility depends on two polarly localized engines, type IV pili pull cells forward, and slime extruding nozzle-like structures appear to push cells forward. The motility behaviour of cells provides evidence that the two engines are localized to opposite poles and that they undergo polarity switching. Several proteins involved in regulating polarity switching have been identified. The cell surface-associated C-signal induces the directed movement of cells into nascent fruiting bodies. Recently, the molecular nature of the C-signal molecule was elucidated and the motility parameters regulated by the C-signal were identified. From the effect of the C-signal on cell behaviour it appears that the C-signal inhibits polarity switching of the two motility engines. This establishes a connection between cell polarity, signalling by an intercellular signal and morphogenetic cell movements during fruiting body formation.     

Limitations of tpi and bg genes sub-genotyping for characterization of human Giardia duodenalis isolates

The intestinal protozoan Giardia duodenalis includes 2 genetically distinct assemblages, A and B, which are responsible for human infections. Little is known so far on the genotypes of G. duodenalis human isolates in France. The present characterization of 19 French clinical isolates was aimed at determining their genotype patterns and associations with clinical symptoms, and in vivo metronidazole resistance, respectively. Based on both triose-phosphate isomerase (tpi) and β-giardin (bg) gene sequences, twelve isolates were identified as assemblage A, and 7 as assemblage B for the 2 gene loci. Sub-genotyping heterogeneities were observed in 15/19 isolates attributed to either A or B assemblage. They include frequent mismatches and intra-assemblage discordances and mixed positions, which were found more frequently in tpi than in bg sequences, and in assemblage B than in assemblage A sequences. No association was found between sub-genotypes, clinical symptoms and metronidazole sensitivity. Present data underline the need for improvements in the standardization of G. duodenalis multilocus genotyping approach for further molecular epidemiologic studies of giardiasis.     

Antifreeze proteins

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice     

Modified vaccinia virus Ankara as antigen delivery system: how can we best use its potential?

Safety-tested modified vaccinia virus Ankara (MVA) has been established as a potent vector system for the development of candidate recombinant vaccines. The versatility of the vector system was recently demonstrated by the rapid production of experimental MVA vaccines for immunization against severe acute respiratory syndrome associated coronavirus. Promising results were also obtained in the delivery of Epstein-Barr virus or human cytomegalovirus antigens and from the clinical testing of MVA vectors for vaccination against immunodeficiency virus, papilloma virus, Plasmodium falciparum or melanoma. Moreover, MVA is considered to be a prime candidate vaccine for safer protection against orthopoxvirus infections. Thus, vector development to challenge dilemmas in vaccinology or immunization against poxvirus bio-threat seems possible, yet the right choice should be made for a most beneficial use.     

Nonstructural HIV proteins as targets for prophylactic or therapeutic vaccines

By the end of 2004, more than 20 HIV-1 vaccine candidates will have entered clinical testing in at least 30 trials worldwide. Almost half of these vaccines include nonstructural HIV-1 gene products. This represents an important innovation in the HIV vaccine field, because until 9 years ago not even preclinical testing in small animal models had been carried out with such immunogens. This review briefly discusses the experimental evidence that provides the rationale for the use of nonstructural HIV-1 gene products as vaccine antigens, and summarizes the current status and the future development of these novel vaccines.     

Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases

HIV undergoes rapid genetic variation; this variation is caused primarily by the enormous number of viruses produced daily in an infected individual. Because of this variation, HIV presents a moving target for drug and vaccine development. The variation within individuals has led to the generation of diverse HIV-1 subtypes, which further complicates the development of effective drugs and vaccines. In general, it is more difficult to hit a moving target than a stationary target. Two broad strategies for hitting a moving target (in this case, HIV replication) are to understand the movement and to aim at the portions that move the least. In the case of anti-HIV drug development, the first option can be addressed by understanding the mechanism(s) of drug resistance and developing drugs that effectively inhibit mutant viruses. The second can be addressed by designing drugs that interact with portions of the viral machinery that are evolutionarily conserved, such as enzyme active sites.     

Characterization of a Mouse Gene (Fbxw6) That Encodes a Homologue of Caenorhabditis elegans SEL-10

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box–WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.     

Rewiring cell signaling: the logic and plasticity of eukaryotic protein circuitry

Living cells rival computers in their ability to process external information and make complex behavioral decisions. Many of these decisions are made by networks of interacting signaling proteins. Ongoing structural, biochemical and cell-based studies have begun to reveal several common principles by which protein components are used to specifically transmit and process information. Recent engineering studies demonstrate that these relatively simple principles can be used to rewire signaling behavior in a process that mimics the evolution of new phenotypic responses.