Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression

Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.     

Reactivation of latent tuberculosis infection in TNF-deficient mice

TNF-deficient mice are highly susceptible to Mycobacterium tuberculosis H37Rv infection. Here we asked whether TNF is required for postinfectious immunity in aerosol-infected mice. Chemotherapy for 4 wk commencing 2 wk postinfection reduced CFU to undetectable levels. While wild-type mice had a slight rise in CFU, but controlled infection upon cessation of chemotherapy, TNF-deficient mice developed reactivation of infection with high bacterial loads in lungs, spleen, and liver, which was fatal within 13-18 wk. The increased susceptibility of TNF-deficient mice was accompanied by diminished recruitment and activation of T cells and macrophages into the lung, with defective granuloma formation and reduced inducible NO synthase expression. Reduced chemokine production in the lung might explain suboptimal recruitment and activation of T cells and uncontrolled infection. Therefore, despite a massive reduction of the mycobacterial load by chemotherapy, TNF-deficient mice were unable to compensate and mount a protective immune response. In conclusion, endogenous TNF is critical to maintain latent tuberculosis infection, and in its absence no specific immunity is generated.     

Phylogeny and the Evolution of the <Emphasis Type="Italic">Amylase</Emphasis> Multigenes in the <Emphasis Type="Italic">Drosophila montium</Emphasis> Species Subgroup

Abstract To investigate the phylogenetic relationships and molecular evolution of α-amylase (Amy) genes in the Drosophila montium species subgroup, we constructed the phylogenetic tree of the Amy genes from 40 species from the montium subgroup. On our tree the sequences of the auraria, kikkawai, and jambulina complexes formed distinct tight clusters. However, there were a few inconsistencies between the clustering pattern of the sequences and taxonomic classification in the kikkawai and jambulina complexes. Sequences of species from other complexes (bocqueti, bakoue, nikananu, and serrata) often did not cluster with their respective taxonomic groups. This suggests that relationships among the Amy genes may be different from those among species due to their particular evolution. Alternatively, the current taxonomy of the investigated species is unreliable. Two types of divergent paralogous Amy genes, the so-called Amy1- and Amy3-type genes, previously identified in the D. kikkawai complex, were common in the montium subgroup, suggesting that the duplication event from which these genes originate is as ancient as the subgroup or it could even predate its differentiation. Thc Amy1-type genes were closer to the Amy genes of D. melanogaster and D. pseudoobscura than to the Amy3-type genes. In the Amy1-type genes, the loss of the ancestral intron occurred independently in the auraria complex and in several Afrotropical species. The GC content at synonymous third codon positions (GC3s) of the Amy1-type genes was higher than that of the Amy3-type genes. Furthermore, the Amy1-type genes had more biased codon usage than the Amy3-type genes. The correlations between GC3s and GC content in the introns (GCi) differed between these two Amy-type genes. These findings suggest that the evolutionary forces that have affected silent sites of the two Amy-type genes in the montium species subgroup may differ.     

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.     

Aphid (Sitobion yakini) investigation suggests thin-walled sieve tubes in barley (Hordeum vulgare) to be more functional than thick-walled sieve tubes

Barley, like most other grasses that have been studied, contains two kinds of sieve tube. The first formed are called thin-walled sieve tubes because of their thin wall compared to the late-formed, and are associated with companion cells. The late-formed are thick-walled sieve tubes, which differentiate next to the metaxylem vessels and lack companion cells. Aphid ( Sitobion yakini (Eastop) feeding was studied using light microscopy to determine if they preferentially feed from thin- or thick-walled sieve tubes in the barley leaf. Penetration of the stylets through the leaf epidermis and mesophyll was largely intercellular, becoming partly intercellular and, partly, intracellular inside the vascular bundle. Sixteen of 19 pairs of stylets (84%), and 293 of 317 (92%) stylet tracks terminated at the thin-walled sieve tubes, suggesting that Sitobion yakini feeds preferentially on the thin-walled sieve tubes which seem to be more attractive to the aphid. These thin-walled sieve tubes are thus probably the most functional in terms of phloem loading and transport.     

Symplastic Solute Transport and Avocado Fruit Development: A Decline in Cytokinin/ABA Ratio is Related to Appearance of the Hass Small Fruit Variant

Studies on the effect of fruit size on endogenous ABA and isopentenyladenine(iP) in developing avocado (Persea americana Mill. cv. Hass)fruit revealed that ABA content was negatively correlated withfruit size whilst the iP/ABA ratio showed a linear relationshipwith increasing size of fruit harvested 226 d after full bloom.The effect of this change in hormone balance on the relationshipbetween symplastic solute transport and appearance of the smallfruit variant was examined following manipulation of the endogenouscytokinin (CK)/ABA ratio. Application of ABA caused seed coatsenescence and retarded fruit growth but these effects wereabsent in fruit treated with equal amounts of ABA plus iP. Thus,the underlying physiological mechanisms associated with ABA-inducedretardation of Hass avocado fruit growth appeared to be inextricablylinked to a decline in CK content and included: diminution ofmesocarp and seed coat plasmodesmatal branching, gating of mesocarpand seed coat plasmodesmata by deposition of electron densematerial in the neck region, abolishment of the electrochemicalgradient between mesocarp and seed coat parenchyma, and arrestof cell-to-cell chemical communication. (Received February 25, 1998; Accepted July 28, 1998)     

Correlation of volatile profiles of twenty mango cultivars with their susceptibilities to mango gall fly infestation

Mango gall fly (Procontarinia matteiana) is an orchard pest that parasitises flush leaves of mango and serious outbreaks may result in reduced fruit yield. The trigger for infestation is unknown, but terpenes emitted by the leaves appear to play a role in attraction. Metabolic profiles of three mango cultivars of varying susceptibility to mango gall fly attack were obtained by headspace profiling using GC-FID and GC-MS analysis. Chemometric models constructed from the data revealed that three terpenes, α- and β-pinene and camphene could be useful as biomarkers for susceptibility. Headspace profiles of twenty other cultivars, naturally exposed to gall fly, were obtained in the same way. Susceptibility or resistance of these cultivars was predicted using the developed orthogonal partial least squares model. Predictive outcomes were thereafter verified by visual examination of the leaves to detect gall formation, an indication of gall fly infestation. The model was found to predict the susceptibility or resistance of 90% of the cultivars accurately. This finding indicates the contributory role of the three terpene biomarkers in mango gall fly interaction and may direct future studies to determine their inter-relationship.     

Phage display as a powerful tool to engineer protease inhibitors

Since its introduction by Georges Smith some 25 years ago, phage display has proved to be a powerful molecular technique for selecting proteins with desired biological properties from huge libraries. Early on, various protease inhibitor scaffolds were displayed at the surface of filamentous phages to select new inhibitors with shifted specificities and enhanced affinities towards one or more target protease(s). The past two decades have seen a number of natural protease inhibitors subjected to phage display, mostly to shift and increase their inhibitory specificity, but also to explore the molecular mechanisms by which they interact with their cognate enzymes with low or very high selectivity. This review focuses on the major uses of phage display in the field of protein protease inhibitors. The exquisite molecular mechanisms by which natural protease inhibitors prevent unwanted or excessive proteolysis in cells and tissues are also examined along with some of the general principles underlying the way phage display is applied to these molecules.