Functional divergence of a heterochromatin‐binding protein during stickleback speciation

Intragenomic conflict, the conflict of interest between different genomic regions within an individual, is proposed as a mechanism driving both the rapid evolution of heterochromatin‐related proteins and the establishment of intrinsic genomic incompatibility between species. Although molecular studies of laboratory model organisms have demonstrated the link between heterochromatin evolution and hybrid abnormalities, we know little about their link in natural systems. Previously, we showed that F₁ hybrids between the Japan Sea stickleback and the Pacific Ocean stickleback show hybrid male sterility and found a region responsible for hybrid male sterility on the X chromosome, but did not identify any candidate genes. In this study, we first screened for genes rapidly evolving under positive selection during the speciation of Japanese sticklebacks to find genes possibly involved in intragenomic conflict. We found that the region responsible for hybrid male sterility contains a rapidly evolving gene encoding a heterochromatin‐binding protein TRIM24B. We conducted biochemical experiments and showed that the binding affinity of TRIM24B to a heterochromatin mark found at centromeres and transposons, histone H4 lysine 20 trimethylation (H4K20me3), is reduced in the Japan Sea stickleback. In addition, mRNA expression levels of Trim24b were different between the Japan Sea and the Pacific Ocean testes. Further expression analysis of genes possibly in the TRIM24B‐regulated pathway showed that some gypsy retrotransposons are overexpressed in the F₁ hybrid testes. We, therefore, demonstrate that a heterochromatin‐binding protein can evolve rapidly under positive selection and functionally diverge during stickleback speciation.     

Construction of Lactobacillus plantarum strain with enhanced L-lysine yield

AIM: To enhance L-lysine secretion in Lactobacillus plantarum. METHODS AND RESULTS: An S-2-aminoethyl-L-cystein (AEC)-resistant mutant of L. plantarum was isolated, and it produced L-lysine at considerably higher level than the parent strain. Aspartokinase in the mutant has been desensitized to feedback inhibition by L-lysine. The nucleotide sequence analysis of thrA2 that codes for aspartokinase in the mutant predicted a substitution of glutamine to histidine at position 421. L-Lysine-insensitive aspartokinase, together with aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase genes, was cloned from L. plantarum DNA to a shuttle vector, pRN14, and the genes were then transformed individually into the AEC-resistant mutant and the parent strain. The overexpression of the genes led to the increase in the activity of enzymes they encode in vitro. However, only the strain overexpressing aspartokinase or dihydrodipicolinate synthase produced more L-lysine. CONCLUSIONS: The desensitization of aspartokinase to L-lysine in L. plantarum led to the overproduction of L-lysine. The overexpression of L-lysine-insensitive aspartokinase or dihydrodipicolinate synthase enhanced L-lysine secretion in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the L-lysine-overproducing strain of L. plantarum in food or feed fermentation may increase the L-lysine content of fermented products.     

Analysis of N-glycan profile of Arabidopsis alg3 cell culture

N-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man₃GlcNAc₂ (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N-glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy.     

Epithelial rests of Malassez express immunoreactivity of TrkA and its distribution is regulated by sensory nerve innervation.

The periodontal ligament is the connective tissue that fills the space between the tooth and its bony socket. It is abundantly innervated by the sensory and sympathetic nerves. We first investigated the immunoreactivity of TrkA, which is a high-affinity receptor of nerve growth factor (NGF), in the periodontal ligament of rats. Immunoreactivity was observed at the epithelial cells in the cervical and furcation regions of the molars. These epithelial cells, which gather together to form clusters or networks, are known as the epithelial rests of Malassez. Immunoreactivity was not observed in other non-neuronal cells, such as osteoblasts, fibroblasts, odontoblasts, cementoblasts, endothelial cells, and/or osteoclasts. On the basis of these findings, we investigated the possible involvement of sensory nerve innervation in the immunoreactivity of the epithelial cells. Denervation of the inferior alveolar nerve resulted in a marked decrease in the distribution area and size of the clusters of immunoreactive cells compared with those of sham-operated rats. These findings suggest that sensory nerve innervation may have a regulatory role in maintenance of the epithelial rests of Malassez expressing TrkA in the periodontal ligament.     

In vitro effects of glycosylated insulin in perfused liver and hindquarter in rats.

Using the perfused liver and hindquarter of the rat, the uptake of glycosylated insulin and its effect on glucose output were investigated. Insulin was glycosylated in ambient high glucose concentration, and glycosylated insulin GI80 (insulin incubated with 0.08% glucose), GI350 (incubated with 0.35% glucose), and GI1000 (incubated with 1% glucose) were prepared. The liver and hindquarter were perfused with nonglycosylated insulin (N-GI) or glycosylated insulin at a concentration of 100 or 1000 microU/ml. There were no significant differences in the fractional uptake of insulin by perfused liver and hindquarter despite glycosylation. Insulin-induced decrement in glucose output was significantly lower in the liver perfused with GI1000 than that in the liver perfused with N-GI, GI80, and GI350 at an insulin concentration of 100 microU/ml. There were no significant differences in insulin-induced decrement in glucose output between the hindquarter perfused with N-GI, GI80, GI350, and GI1000. These results suggest that when insulin (100 microU/ml) is incubated with a markedly elevated concentration of glucose (1000 mg/dl) its biological activity is reduced in the liver, but not in the hindquarter.     

Glypican-3, overexpressed specifically in human hepatocellular carcinoma,is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.     

Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.

Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.     

Decrease in Activity of Glutathione Reductase Enhances Paraquat Sensitivity in Transgenic Nicotiana tabacum

Transgenic tobacco (Nicotiana tabacum L. cv SR1) with decreased activity of glutathione reductase exhibited enhanced sensitivity to paraquat in the light as evaluated by chlorophyll destruction and electrolyte leakage from leaf discs. This result indicates the involvement of glutathione reductase in the tolerance of plants to photooxidative stress caused by the herbicide.