Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni²⁺-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.     

Phylogenetic relationships ofPelvetia andPelvetiopsis (Phaeophyceae) based on small subunit ribosomal DNA sequences

Nucleotide sequences of the small-subunit (SSU) ribosomal DNA were determined forPelvetia babingtonii, P. canaliculate, Pelvetiopsis limitata, andAscophyllum nodosum in the family Fucaceae. A total of 1755 positions were aligned for the whole sequence. The positional differences in the primary structure among the taxa ranged from 16 to 30 nucleotide changes in pairwise comparisons. There was a minimum divergence betweenPs. limitata andP. babingtonii while a maximum betweenPs. limitata andP. canaliculata. The SSU rDNA trees showed that the genusPelvetia was not monophyletic and the genusPelvetiopsis was not closely related toPelvetia. Our results suggest that the taxonomic revision of the genusPelvetia as well as the family Fucaceae is needed based on detailed morphological observations.     

Internal ribosome-binding site directs expression of parathyroid hormone analogue (8-84) in Escherichia coli

Expression of the human parathyroid hormone (PTH) gene in E. coli yielded intact PTH and PTH-(8-84). To determine if PTH-(8-84) is the result of a competing translation initiated from methionine codon-8 or degradation of the intact PTH, twelve new gene constructs with or without an internal ribosome-binding site (iRBS) in the PTH-(1-5) region were prepared via substitution with degenerate codons. Expression of constructs without iRBS produced only intact PTH. Constructs with weak iRBS, including one that resembles the cDNA sequence, yielded PTH-(8-84) as a minor product. In contrast, constructs with strong iRBS produced predominantly or exclusively this shorter analogue.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EII^Man) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EII^Man showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EII^Glc). Similar homology was shown between the C-terminal sequence of EII^Man and the E. coli glucose-specific enzyme III (EIII^Glc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EII^Man contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIII^Glc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

The effect of multi-frequency whole-body vibration on night-shifted mouse model

The circadian rhythm controls several biological activities; therefore, a disorganized circadian rhythm may cause fatal health problems. The aim of this study was to assess the effects of circadian rhythm disturbances induced by simulated night shift activities on the abdominal adipose tissue, bone microstructures and muscle volume in the tibiae of mice. Moreover, we evaluated the effects of multi-frequency whole-body vibration as a countermeasure against the consequences of circadian rhythm disturbances. Twenty-four 5-week-old C57BL/6J male mice were equally assigned to three groups: the normal group (Nor), night shift group (NS), and night shift with multi-frequency whole-body vibration group (NS + V). The NS and NS + V groups were exposed to circadian rhythm disturbances for 4 weeks with 3-day intervals by changing the day and night cycle based on 7 o’clock. After 4 weeks, morphological changes in the adipose tissue, bone microstructures and muscle volume in the tibiae were evaluated from three-dimensional images using in vivo micro-computed tomography. As a result, the volume of the abdominal adipose tissue was significantly higher in the NS than in the Nor and NS + V groups. Also, the microstructures of the tibia were more enhanced in the NS + V than the NS group. The volume of tibial muscle was increased in all groups, while there were no significant changes in muscle volume. From these results, we can conclude that circadian rhythm disturbances induced by night shift activities may reduce bone condition and increase the accumulation of abdominal adipose tissue and these negative effects may be prevented or improved through applying multi-frequency whole-body vibration.

     

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer

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Ascorbic Acid Ameliorates Gestational Lead Exposure-Induced Developmental Alteration in GAD67 and c-Kit Expression in the Rat Cerebellar Cortex

In the present study, we investigated the effects of ascorbic acid on lead-exposed developing cerebellum. Female rats were divided into the following three groups: control (distilled water), lead (0.2% lead acetate), and lead plus ascorbic acid (100 mg/kg/day, 10% solution). To evaluate the effect of lead exposure and ascorbic acid treatment accurately on the cerebellar development for the gestational period, we halted further treatment with lead and ascorbic acid in the dams after delivery of the pups. Although the ascorbic acid slightly decreased the lead level in pups, lead level was still high in the group treated with lead plus ascorbic acid group compared with the control group. The blood lead levels indicated that the ascorbic acid could facilitate both the excretion and transfer of lead from a dam to its pups via milk. At postnatal day 21, lead exposure significantly reduced the number of Purkinje cells in the cerebellar cortex of pups. Additionally, lead treatment induced degenerative changes such as reduction of glutamic acid decarboxylase (GAD67) and c-kit expressions are observed in the developing cerebellar cortex. In the cerebellum of the pups from the lead plus ascorbic acid group, reduction of the number of Purkinje cells, GAD67 expression, and c-kit immunopositivity were remarkably restored compared with the lead group. Our present results suggested that ascorbic acid treatment to lead-exposed dam exerted protective effects on the developing cerebellum against lead-induced neurotoxicity.     

Immuno-electron microscopy reveals that the excitotoxin quinolinate is associated with the plasma membrane in human peripheral blood monocytes/macrophages

Quinolinate (QUIN), a tryptophan-derived excitotoxin, was localized ultrastructurally in human peripheral blood monocytes/macrophages (M?) by immuno-electron microscopy. A combined carbodiimide/glutaraldehyde/paraformaldehyde-based fixation procedure was developed for optimal retention of QUIN in the cell as well as minimal loss of ultrastructure; a silver-enhanced colloidal gold detection system was used for electron-microscopic analysis. Gold particles representing QUIN immunoreactivity were associated with the inner side of the plasma membrane in normal M?. The number of gold particles increased significantly when QUIN levels were elevated by treatment with its precursor kynurenine, but location of the gold particles remained essentially the same under this condition. Treatment with interferon-γ increased the number of Golgi bodies, vacuoles and pseudopodia, reflecting the activated state of the cell. Significantly increased numbers of gold particles representing QUIN were detectable in approximately the same location as in the case of kynurenine treatment. Combined treatment with kynurenine and interferon-γ maximally increased the number of gold particles at the periphery of the cell. The pseudopodia were intensely stained with gold particles, while they were not detectable in the inner part of the cytoplasm or in any other organelle even under this activated condition. The significance of the specific location of QUIN revealed in the present study and its relation to the release and subsequent actions of QUIN are discussed. Received: 30 May 1996 / Accepted: 22 May 1997