Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

Geminivirus-based vectors for gene silencing in Arabidopsis

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.     

Epididymal cribriform hyperplasia with nuclear atypia in p53 homozygous knockout mice on a mixed 129/Sv-FVB/N background

Epididymal cribriform hyperplasia (ECH) is a variant of normal epididymal histologic features in men, and has also been reported in rats, mice, dogs, cats, and bulls. The epididymal change has been associated with aging, testicular atrophy, cryptorchidism, and germ cell tumors. Epididymal cribriform hyperplasia was observed in p53 homozygous knockout mice on a mixed 129/Sv-FVB/N background, but not in wild-type or heterozygous mice. The aim of the study reported here was to determine the prevalence and characterize the morphologic, immunohistochemical, and ultrastructural features of ECH in these mice. Epididymal cribriform hyperplasia was present in 88% (72/82) of male mice ranging in age from seven to 65 weeks. The lesion was characterized microscopically by epithelial cells with atypical hyperchromatic nuclei, vacuolization, intratubular lumina formation, infrequent apoptosis, and rare mitotic figures. In contrast to germ cells, the cells of ECH did not express alpha-fetoprotein, carcinoembryonic antigen, or S-100. Ultrastructurally, the cells were pleomorphic with stereocilia at their apical borders and within intratubular lumina, and were supported by a basement membrane. Although 14% (10/72) of mice had concomitant testicular neoplasia, ECH did not appear to be a preneoplastic change. Investigators using these mice for modeling human disease should be aware of the background prevalence of this lesion.     

Pseudostructural inhibitors of G protein signaling during development

Heterotrimeric G proteins mediate signal transduction pathways to control development in fungal, plant, and animal cells. A recent study in the July issue of Molecular Cell identifies three proteins that, while not displaying sequence similarity to G protein subunits, appear to act as structural mimics of a Gbetagamma dimer to negatively regulate pseudohyphal growth in budding yeast.     

Effect of massive sympathetic nervous system activation on coronary blood flow and myocardial energy pool

Our previous work indicates that myocardial ischemia could be the mechanism responsible for the left ventricular (LV) dysfunction that frequently develops after massive sympathetic nervous system (SNS) activation. In this study, coronary blood flow (CBF) and myocardial ATP, creatine phosphate, and lactate concentrations were measured after massively activating the SNS of anesthetized rabbits with an intracisternal injection of veratrine. CBF was measured at time 0 (baseline), and at 2, 10, and 20 min after SNS activation in one group, and at 0, 45, 90, and 150 min in a second group. Myocardial ATP, creatine phosphate, and lactate were measured at 0, 2, 10, 20, 90, and 150 min in separate groups of rabbits. SNS activation caused LV dysfunction in approximately 60% of the rabbits. SNS-related increases in CBF kept pace with the increases in myocardial energy demand as determined from the systolic pressure-heart rate product. The subendocardial-to-subepicardial blood flow ratio did not change significantly. Myocardial creatine phosphate concentration was depressed 2 min after SNS activation and remained depressed for at least 20 min. ATP fell continuously and was significantly lower than baseline by 20 min. Tissue lactate concentration was elevated at this time. By 90 min, the concentrations of all three metabolites had recovered. These results indicate that myocardial high-energy phosphate compounds fall after massive SNS activation, but ischemia does not appear to be the underlying mechanism.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Dementia,the fate of brain? Neuropathological point of viewLe destin du cerveau ? Un point de vue neuropathologique sur les démences.

The prevalence of dementia dramatically increases during ageing, and this puts a serious strain on the optimism brought by the continuous increase in life expectancy observed in most industrialised countries. Diseases that produce dementia are numerous, and the cognitive deficit results from lesions of various regions and from different mechanisms. This modulates the possible prediction, prevention and cure of dementia. Emphasis is put on the necessity of, and prerequisite for, efficient research in the field of dementia. Three paradigmatic dementing disorders are reviewed. Subacute spongiform encephalopathies (prion diseases) constitute a biological enigma and a public health concern. In Alzheimer's disease and vascular or mixed dementia, the clinical diagnosis is still imperfect, and this hinders research. Distinguishing and accurately identifying the various types of dementia is essential for understanding their mechanism and for developing efficient therapeutic strategies, preventive and curative. For such objectives, the study of human brain tissue will remain mandatory until non-invasive markers and additional models are available. Ethical reasons banish the use of cerebral biopsy and favour the promotion of autopsy.     

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.