Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.     

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

BackgroundMutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant—associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.MethodsWe investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26^f/fP0Cre).ResultsIn ultrastructural observations of Cx26^f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26^f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26^f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.ConclusionsThe deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.     

Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.     

Plasma free amino acid profiling of five types of cancer patients and its application for early detection

Background

Recently, rapid advances have been made in metabolomics-based, easy-to-use early cancer detection methods using blood samples. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. Furthermore, PFAA profiles are known to be influenced by specific diseases, including cancers. Therefore, the purpose of the present study was to determine the characteristics of the PFAA profiles in cancer patients and the possibility of using this information for early detection.

Methods and Findings

Plasma samples were collected from approximately 200 patients from multiple institutes, each diagnosed with one of the following five types of cancer: lung, gastric, colorectal, breast, or prostate cancer. Patients were compared to gender- and age- matched controls also used in this study. The PFAA levels were measured using high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS). Univariate analysis revealed significant differences in the PFAA profiles between the controls and the patients with any of the five types of cancer listed above, even those with asymptomatic early-stage disease. Furthermore, multivariate analysis clearly discriminated the cancer patients from the controls in terms of the area under the receiver-operator characteristics curve (AUC of ROC >0.75 for each cancer), regardless of cancer stage. Because this study was designed as case-control study, further investigations, including model construction and validation using cohorts with larger sample sizes, are necessary to determine the usefulness of PFAA profiling.

Conclusions

These findings suggest that PFAA profiling has great potential for improving cancer screening and diagnosis and understanding disease pathogenesis. PFAA profiles can also be used to determine various disease diagnoses from a single blood sample, which involves a relatively simple plasma assay and imposes a lower physical burden on subjects when compared to existing screening methods.     

Identification and Functional Characterization of the First Nucleobase Transporter in Mammals: IMPLICATION IN THE SPECIES DIFFERENCE IN THE INTESTINAL ABSORPTION MECHANISM OF NUCLEOBASES AND THEIR ANALOGS BETWEEN HIGHER PRIMATES AND OTHER MAMMALS*

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 μm. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.     

The human HYMAI/PLAGL1 differentially methylated region acts as an imprint control region in mice

Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain.