Identification of a novel mutation in the ß-globin gene 3' untranslated region [+1,506 (A>C)] in a Japanese male with a heterozygous ß-thalassemia phenotype.

ß-Thalassemia (ß-thal) is characterized by the absent or reduced production of ß-globin chains. The precise molecular lesion that causes decreased ß-globin synthesis in ß(+)-thal is difficult to predict when mutations occur in the locus control region (LCR), the promoter, the introns or 3' untranslated regions (3'UTRs). Among them, the role of the 3'UTR of ß-globin gene in mRNA stability is poorly understood, mainly due to very few cases that have mutations in this region. So far, only three mutations have been reported in the 3'UTR of ß-globin gene. Although, it is speculated that some of these reported mutations could be associated with mRNA stability, the precise molecular basis still remains unclear. We report here a novel mutation in the ß-globin gene 3'UTR [+1,506 (A>C)] in a 31-year-old Japanese male with hematological parameters suggestive of heterozygous ß-thal. Further functional studies on this novel mutation reported here, may help in understanding of the regulation and expression of the ß-globin gene and its products.

The +1,506 (A>C) mutation in the 3' untranslated region affects ß-globin expression.

The 3' untranslated region (3'UTR) is known to be important to mRNA stability but the stabilization mechanism on the ß-globin gene is not fully elucidated. We speculated in our previous report that +1,506 (A>C) mutation (HGVS nomenclature: *32A>C) on the ß-globin 3'UTR causes ß-thalassemia (ß-thal) in order to destabilize the mRNA. To investigate further, we studied the expression efficiency for the mutation with a luciferase assay. We made recombinant pGL4.74 vectors in which the luciferase 3'UTR was replaced with the wild-type and mutant 3'UTR of the ß-globin gene. For a comparison experiment, recombinant vectors were made not only for this mutation but also six other mutations in the ß-globin 3'UTR which bring about ß-thal or affect mRNA stability. The +1,506 mutation led to a 30.0% lower protein expression than normal in this assay. We concluded that this mutation destabilizes mRNA and consequently decreases the ß-globin amount to finally cause ß-thal. Our study highlights the crucial area of ß-globin 3'UTR for protein expression.

GaInAsP/InP lateral-current-injection distributed feedback laser with a-Si surface grating.

We fabricated a novel lateral-current-injection-type distributed feedback (DFB) laser with amorphous-Si (a-Si) surface grating as a step to realize membrane lasers. This laser consists of a thin GaInAsP core layer grown on a semi-insulating InP substrate and a 30-nm-thick a-Si surface layer for DFB grating. Under a room-temperature continuous-wave condition, a low threshold current of 7.0 mA and high efficiency of 43% from the front facet were obtained for a 2.0-µm stripe width and 300-µm cavity length. A small-signal modulation bandwidth of 4.8 GHz was obtained at a bias current of 30 mA.

A Novel Cognitive Function Scale Using Functional Near-Infrared Spectroscopy for Evaluating Cognitive Dysfunction.

BACKGROUND: Maintaining cognitive function is integral to a healthy social life in the aged. Although neuropsychological tests and brain imaging methods can assess cognitive dysfunction, these techniques are subjective, psychologically burdensome, and cannot be conducted easily. OBJECTIVE: We sought to develop an objective, low-burden novel cognitive function scale based on functional near-infrared spectroscopy (fNIRS) of hemodynamic changes in the cerebral cortex during daily task performance. METHODS: A total of 63 participants (aged 60-80 years) identified as non-dementia controls (NDC) or with mild cognitive impairment (MCI) were recruited and randomly assigned to training and test data sets. Explanatory variables were hemodynamic responses during low-burden sensory and simple tasks without higher-order brain functioning. RESULTS: A logistic regression analysis of the fNIRS index in NDCs and MCI patients revealed area under the curve, sensitivity, specificity, and holdout results of 0.98, 94%, 88%, and 62% respectively. Correlation between fNIRS index and MCI odds showed positive linearity (R2 = 0.96). CONCLUSION: Positive correlation between the fNIRS index and MCI odds indicated effectiveness of this fNIRS measurement. Although additional experiments are necessary, the fNIRS index representing degree of cognitive decline can be an onsite monitoring tool to assess cognitive status.

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants.

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M(2) seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.

Lateral current injection GaInAsP/InP laser on semi-insulating substrate for membrane-based photonic circuits.

A room-temperature pulsed operation was demonstrated using lateral current injection-type lasers composed of a 400-nm-thick GaInAsP core layer with compressively strained 5 quantum wells. A threshold current of 105 mA and corresponding density of 1.3 kA/cm(2) (260 A/cm(2) per well) were obtained with the stripe width of 5.4 microm and the cavity length of 1.47 mm. A fundamental transverse mode operation was obtained with the narrower stripe device of 2.0 microm and the cavity length of 805 microm, while the threshold current and corresponding density were 49 mA and 3.0 kA/cm(2), respectively.

Serum levels of proteins involved in amyloid-ß clearance are related to cognitive decline and neuroimaging changes in mild cognitive impairment.

INTRODUCTION: Amyloid-ß (Aß) clearance is important for damage prevention in Alzheimer's disease. We investigated the utility of Aß clearance proteins as biomarkers for mild cognitive impairment (MCI). METHODS: Serum apolipoprotein (apo) A-I, compliment protein C3 (C3), transthyretin, and cholesterol levels were measured in 273 subjects, and we analyzed the relationship between these levels and brain atrophy and cerebral blood flow in 63 clinically diagnosed mild cognitive impairment, Alzheimer's disease, and nondemented disease control subjects. RESULTS: ApoA-I and transthyretin levels and the active form of C3:native form of C3 ratio achieved an area under the curve of 0.89 (sensitivity: 83%, specificity: 90%) for detecting late mild cognitive impairment. Atrophy was associated with decreased apoA-I and high-density lipoprotein levels. Subjects with reduced cerebral blood flow had lower levels of active form of C3, apoA-I, high-density lipoprotein, and total cholesterol. Low native form of C3 and high active form of C3 levels were found in the hippocampi of patients with Alzheimer's disease. DISCUSSION: Aß clearance proteins in the serum are potential biomarkers for mild cognitive impairment evaluation.

Mass measuring instrument for use under microgravity conditions.

A prototype instrument for measuring astronaut body mass under microgravity conditions has been developed and its performance was evaluated by parabolic flight tests. The instrument, which is the space scale, is applied as follows. Connect the subject astronaut to the space scale with a rubber cord. Use a force transducer to measure the force acting on the subject and an optical interferometer to measure the velocity of the subject. The subject's mass is calculated as the impulse divided by the velocity change, i.e., M=integral Fdt/delta v. Parabolic flight by using a jet aircraft produces a zero-gravity condition lasting approximately 20 s. The performance of the prototype space scale was evaluated during such a flight by measuring the mass of a sample object.

Dai-Kenchu-To, a Herbal Medicine, Attenuates Colorectal Distention-induced Visceromotor Responses in Rats.

BACKGROUND/AIMS: Dai-kenchu-to (DKT), a traditional Japanese herbal medicine, is known to increase gastrointestinal motility and improve ileal function. We tested our hypotheses that (1) pretreatment with DKT would block the colorectal distention-induced visceromotor response in rats, and (2) pretreatment with DKT would attenuate colorectal distention-induced adrenocorticotropic hormone (ACTH) release and anxiety-related behavior. METHODS: Rats were pretreated with vehicle or DKT (300 mg/kg/5 mL, per os). Visceromotor responses were analyzed using electromyography in response to colorectal distention (10, 20, 40, 60, and 80 mmHg for 20 seconds at 3-minutes intervals). Anxiety-related behavior was measured during exposure to an elevated-plus maze after colorectal distention. Plasma ACTH and serum corticosterone levels were measured after exposure to the elevated-plus maze. RESULTS: Colorectal distention produced robust contractions of the abdominal musculature, graded according to stimulus intensity, in vehicle-treated rats. At 40, 60, and 80 mmHg of colorectal distention, the visceromotor responses of DKT-treated rats was significantly lower than that of vehicle-treated rats. At 80 mmHg, the amplitude was suppressed to approximately one-third in DKT-treated rats, compared with that in vehicle-treated rats. Smooth muscle compliance and the velocity of accommodation to 60 mmHg of stretching did not significantly differ between the vehicle-treated and DKT-treated rats. Similarly, the DKT did not influence colorectal distention-induced ACTH release, corticosterone levels, or anxiety-related behavior in rats. CONCLUSIONS: Our results suggest that DKT attenuates the colorectal distention-induced visceromotor responses, without increasing smooth muscle compliance, ACTH release or anxiety-related behavior in rats.

Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen.

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.

Cloning of a cDNA encoding the Gly m Bd 28K precursor and its vacuole transport in tobacco BY2 suspension-cultured cells.

Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.

Lanthanum carbonate for hyperphosphatemia in patients on peritoneal dialysis.

BACKGROUND: The efficacy of the phosphate binder lanthanum carbonate has been demonstrated for hemodialysis patients, but no studies have focused on patients undergoing continuous ambulatory peritoneal dialysis (CAPD). We evaluated whether lanthanum carbonate could control phosphate levels in patients on CAPD. ♢ METHODS: In this 48-week open-label prospective study, 28 patients on CAPD with a phosphate level of 6 mg/dL or greater were given lanthanum carbonate titrated from 750 mg to 2250 mg daily to achieve a target serum phosphate level of less than 6 mg/dL. The primary efficacy endpoint was reduction of serum phosphate to less than 6 mg/dL. Serum levels of calcium and parathyroid hormone were also evaluated, as were the Ca×P product and adverse effects. ♢ RESULTS: From week 4 to the end of the study at week 48, we observed a significant reduction of serum phosphate to 5.25 ± 0.97 mg/dL from 6.88 ± 1.06 mg/dL at study start (p < 0.01). At the end of the study, 78.6% of participants had achieved the target of less than 6 mg/dL. Because no change of serum calcium occurred, the Ca×P product declined significantly during the study. Intact parathyroid hormone declined gradually over the study period, but the change had not reached significance at the end of the study (p = 0.11). The mean final dose of lanthanum carbonate was 946 mg daily. The only adverse effect reported was mild nausea in 1 patient. ♢ CONCLUSIONS: Lanthanum carbonate is an effective phosphate binder that can control serum phosphate and Ca×P product in CAPD patients with hyperphosphatemia. Lanthanum carbonate was well tolerated in our population.

Aminolytic reaction catalyzed by D-stereospecific amidohydrolases from Streptomyces spp.

From investigation of 2000 soil isolates, we identified two serine-type amidohydrolases that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Streptomyces species 82F2 and 83D12. The enzymes, redesignated as 82F2-DAP and 83D12-DAP, were purified for homogeneity and characterized. Each enzyme had molecular mass of approximately 40 kDa, and each showed moderate stability with respect to temperature and pH. Among hydrolytic activities toward d-aminoacyl-pNAs, the enzymes showed strict specificity toward D-Phe-pNA, but showed broad specificity toward D-aminoacyl esters. The specific activity for D-Phe-pNA hydrolysis of 82F2-DAP was ten-fold higher than that of 83D12-DAP. As a second function, each enzyme showed peptide bond formation activity by its function of aminolysis reaction. Based on results of D-Phe-D-Phe synthesis under various conditions, we propose a reaction mechanism for D-Phe-D-Phe production. Furthermore, the enzymes exhibited peptide elongation activity, producing oligo homopeptide in a one-pot reaction. We cloned the genes encoding each enzyme, which revealed that the primary structure of each enzyme showed 30-60% identity with those of peptidases belonging to the clan SE, S12 peptidase family categorized as serine peptidase with d-stereospecificity.

Proteomic analysis identifies proteins that continue to grow hepatic stem-like cells without differentiation.

To understand the molecular mechanism underlying vigorous proliferative activity of hepatic stem-like (HSL) cells, we performed two-dimensional electrophoresis to identify the proteins statistically more abundant in rapidly growing undifferentiated HSL cells than in sodium butyrate-treated differentiated HSL cells. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Mascot search identified 6 proteins including prohibitin, vimentin, ezrin, annexin A3, acidic ribosomal phosphoprotein P0 and Grp75. Prohibitin and vimentin control the mitogen-activated protein (MAP) kinase pathway. Ezrin is phosphorylated by various protein-tyrosine kinases and modulates interactions between cytoskeletal and membrane proteins. Annexin A3 has a role in DNA synthesis. Acidic ribosomal phosphoprotein P0 and Grp75 play in protein synthesis. These results suggest that the proteins related to the MAP kinase cascade had some role in continuous proliferation of HSL cells without differentiation.

Corticotropin-releasing hormone receptor 1 antagonist blocks brain-gut activation induced by colonic distention in rats.

BACKGROUND & AIMS: The corticotropin-releasing hormone receptor 1 mediates stress-induced changes in colonic motor activity and emotion. We tested the hypothesis that pretreatment with JTC-017, a specific corticotropin-releasing hormone receptor 1 antagonist, blocks colorectal distention-induced hippocampal noradrenaline release and visceral perception in rats. We also investigated whether pretreatment with JTC-017 blocks acute or chronic colorectal distention-induced adrenocorticotropic hormone release, anxiety, and stress-induced changes in colonic motility. METHODS: Rats were pretreated intrahippocampally with alpha-helical corticotropin-releasing hormone (1.25 microg/kg; vehicle), a nonspecific corticotropin-releasing hormone receptor antagonist, or intraperitoneally with JTC-017 (10 mg/kg). Hippocampal noradrenaline release after microdialysis and the frequency of abdominal contractions were measured in response to acute colorectal distention. Plasma adrenocorticotropic hormone levels, anxiety-related behavior, and stress-induced changes in colonic motility were evaluated after acute or chronic colorectal distention followed by exposure to an elevated plus maze. RESULTS: Administration of alpha-helical corticotropin-releasing hormone or JTC-017 significantly attenuated hippocampal noradrenaline release and reduced the frequency of abdominal contractions induced by acute distention. In addition, JTC-017 significantly reduced plasma adrenocorticotropic hormone and anxiety after acute distention. After chronic distention, changes in plasma adrenocorticotropic hormone and anxiety were not significant because of habituation. In contrast, a significant increase in fecal pellet output during the elevated plus maze was observed after chronic distention. This increase in fecal pellet output was blocked by pretreatment with JTC-017. CONCLUSIONS: Our results suggest that JTC-017, a specific corticotropin-releasing hormone receptor 1 antagonist, attenuates hippocampal noradrenaline release, visceral perception, adrenocorticotropic hormone release, and anxiety after acute colorectal distention in rats. In addition, JTC-017 blocks stress-induced changes in colonic motility after chronic colorectal distention in rats.