Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

Role of Tom5 in maintaining the structural stability of the TOM complex of mitochondria

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.     

Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells

The stomach was reported to synthesize and secrete leptin mainly in the gastric lumen. Gastric leptin release is markedly increased after food intake, by vagal cholinergic stimulation and by cholecystokinin and secretin. Here we show that human gastric MKN-74 cells produce leptin that increases upon challenge with cholecystokinin, insulin, glucocorticoids and all-trans retinoic acid through activation of the leptin gene promoter. In addition, we demonstrate that forskolin and BRL37344 which increased cAMP levels, fail to affect the activity of leptin gene promoter in MKN74 expressing beta(3)-adrenoceptor cells but, induce a 2-fold decrease in this activity in adipose 3T3-L1 cells. These data described for the first time, similarities and more interestingly, differences in the regulation of the leptin gene promoter in gastric cells as compared to adipocytes.     

Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates

Since the process of becoming dead genes or pseudogenes (pseudogenization) is irreversible and can occur rather rapidly under certain environmental circumstances, it is one plausible determinant for characterizing species specificity. To test this evolutionary hypothesis, we analyzed the tempo and mode of duplication and pseudogenization of bitter taste receptor (T2R) genes in humans as well as in 12 nonhuman primates. The results show that primates have accumulated more pseudogenes than mice after their separation from the common ancestor and that lineage-specific pseudogenization becomes more conspicuous in humans than in nonhuman primates. Although positive selection has operated on some amino acids in extracellular domains, functional constraints against T2R genes are more relaxed in primates than in mice and this trend has culminated in the rapid deterioration of the bitter-tasting capability in humans. Since T2R molecules play an important role in avoiding generally bitter toxic and harmful substances, substantial modification of the T2R gene repertoire is likely to reflect different responses to changes in the environment and to result from species-specific food preference during primate evolution.     

Chondromodulin-I and tenomodulin: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues

In tissues and/or organs of mesenchymal origin, the vasculature is usually well developed. However, there are certain hypovascular tissues that exhibit powerful anti-angiogenic resistance, implying the presence of tissue-type specific inhibitors of angiogenesis. Hyaline cartilage is one example, and several anti-angiogenic factors have been purified from cartilage. We previously identified chondromodulin-I (ChM-I) as a tissue-specific inhibitor of angiogenesis in fetal bovine cartilage. ChM-I is specifically expressed in the avascular regions of the growth-plate and cartilaginous bone rudiments in embryos. Recently, we cloned a novel type II transmembrane protein, tenomodulin (TeM), having a domain homologous to ChM-I at its C-terminus. TeM turned out to be expressed specifically in other hypovascular structures in the mesenchyme, such as the epimysium, tendon, and ligaments. In this overview, we discuss the structural characteristics of this class of anti-angiogenic molecules and their pathophysiological role in the control of vascularity.     

High level of resistance to metronidazole and clarithromycin in<Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolated from pediatric patients in Poland (1997–2001)

Resistance to metronidazole (Met), clarithromycin (Cla) and amoxycillin (Amo) was examined using H. pylori isolates from child patients before and after treatment in the period 1997-2001. The rate of Met and Cla resistance before treatment was 35.2 and 8.6%, respectively. Six weeks after treatment 48.4% of the isolated strains were resistant to Met and 17.6% to Cla. The highest rate of resistance to both antibiotics was determined in 2001 (before treatment, 46 and 15%, respectively, and after treatment, 57.8 and 26.3%, respectively). All the strains were susceptible to Amo. Strains resistant to Met were detected more frequently in girls than in boys.     

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.     

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the β-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.