Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Urinary iodide excretion measured with an iodide-selective ion electrode: studies on normal subjects of varying ages and patients with thyroid diseases

As a part of studies concerning clinical application of the measurement of urinary iodide with an iodide-selective ion electrode, we report here the reference values for the iodide content or concentration in morning spot urine specimens from normal subjects of varying ages and studies with patients with thyroid diseases in Japan. The number distribution of the iodide content or concentration in the morning specimens appeared logarithmic normal in adults, children and infants. Normal ranges found in 95 per cent of populations of healthy subjects were 5.3 to 62.0 mumoles/g Creatinine (Cr) for adults, 5.3 to 42 mumoles/g Cr for children, and 1.9 to 56 microM for infants, respectively. Urinary iodide concentrations in breast-fed infants varied widely compared with those in bottle-fed infants. Mean values were 16.4 microM for breast-fed infants and 8.6 microM for bottle-fed infants, and they were not statistically different. Urine samples from the infants with transient hypothyroidism, who had undergone amniofetography showed extraordinarily high iodide concentrations, even though they were measured at 20th, 29th and 30th days after birth. Although urinary iodide excretion in patients with simple goiter was within normal limits, the mean was statistically lower than that in normal controls (p less than 0.001). Because of the simplicity and rapidity of the electrode method, we strongly recommend it for use in examining iodide excretion in patients with various thyroid diseases.     

Role of Lung Function Genes in the Development of Asthma

Although our previous GWAS failed to identify SNPs associated with pulmonary function at the level of genomewide significance, it did show that the heritability for FEV₁/FVC was 41.6% in a Japanese population, suggesting that the heritability of pulmonary function traits can be explained by the additive effects of multiple common SNPs. In addition, our previous study indicated that pulmonary function genes identified in previous GWASs in non-Japanese populations accounted for 4.3% to 12.0% of the entire estimated heritability of FEV₁/FVC in a Japanese population. Therefore, given that many loci with individual weak effects may contribute to asthma risk, in this study, we created a quantitative score of genetic load based on 16 SNPs implicated in lower lung function in both Japanese and non-Japanese populations. This genetic risk score (GRS) for lower FEV₁/FVC was consistently associated with the onset of asthma (P = 9.6 × 10⁻⁴) in 2 independent Japanese populations as well as with the onset of COPD (P = 0.042). Clustering of asthma patients based on GRS levels indicated that an increased GRS may be responsible for the development of a particular phenotype of asthma characterized by early onset, atopy, and severer airflow obstruction.     

Long-Term Colonization by blaCTX-M-Harboring Escherichia coli in Healthy Japanese People Engaged in Food Handling

The actual state of intestinal long-term colonization by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 μg/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla_CTX-M were repeatedly recovered from 11 of the 13 carriers of bla_CTX-M-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla_CTX-M-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla_CTX-M-15 or bla_CTX-M-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.     

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.     

The novel RacE-binding protein GflB sharpens Ras activity at the leading edge of migrating cells

Directional sensing, a process in which cells convert an external chemical gradient into internal signaling events, is essential in chemotaxis. We previously showed that a Rho GTPase, RacE, regulates gradient sensing in Dictyostelium cells. Here, using affinity purification and mass spectrometry, we identify a novel RacE-binding protein, GflB, which contains a Ras GEF domain and a Rho GAP domain. Using biochemical and gene knockout approaches, we show that GflB balances the activation of Ras and Rho GTPases, which enables cells to precisely orient signaling events toward higher concentrations of chemoattractants. Furthermore, we find that GflB is located at the leading edge of migrating cells, and this localization is regulated by the actin cytoskeleton and phosphatidylserine. Our findings provide a new molecular mechanism that connects directional sensing and morphological polarization.     

Localization of the gene responsible for Peutz-Jeghers syndrome within a 6-cM region of chromosome 19p13.3

Patients with Peutz-Jeghers syndrome (PJS), an autosomal dominant disease characterized by hamartomatous polyposis of the gastrointestinal tract, are thought to be predisposed to malignancies of the digestive tract, genital tract, and other organs. Using microsatellite markers on chromosome 19p, we have closely defined the region containing the gene responsible for this disorder through linkage analysis in seven affected families. The lack of obligate recombinants at two of these loci, D19S883 and D19S878, with maximum LOD scores of 2.88 and 3.75, confirmed the localization of the PJS locus to chromosome 19. Furthermore, haplotype analysis placed the PJS locus within a 6-cM telomeric region of chromosome 19p, between D19S886 and D19S565. Received: 18 August 1997 / Accepted: 5 November 1997