Non-isotopic microtitre plate-based assay for detecting products of polymerase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus.

A non-isotopic microtitre plate-based assay method was devised for detection of products of the polymerase chain reaction. This assay involves affinity immobilization of the biotinylated amplification products in microtitre plate wells and their fluorescence detection by their hybridization with an oligonucleotide probe linked to alkaline phosphatase. An advantage of this procedure is that the immobilization and hybridization are carried out simultaneously in the wells, thus shortening the assay time. The assay method was applied to the detection of the tdh gene of Vibrio parahaemolyticus. Seven copies of the target chromosome could be detected in about 45 min after 35 cycles of amplification.     

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.     

Evidence for nonclonal hematopoietic progenitor cell populations in bone marrow of patients with myelodysplastic syndromes

Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS) was analyzed by X-chromosome inactivation pattern using polymerase chain reaction (PCR). Five female patients were included in this study; two with refractory anemia (RA) and three with RA with excess blasts (RAEB). They were heterozygous for BstXI restriction fragment length polymorphisms (RFLP) of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each patient, erythroid and nonerythroid colonies, grown in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibited no remarkable difference in clonal constitution. Two patients showed only one methylation pattern, suggesting the monoclonal origin of hematopoietic progenitor cells. Colonies of two other patients exhibited predominant and minor methylation patterns in PGK gene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony- stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells can persist in a substantial number of MDS patients.     

Impaired consciousness caused by a metastatic adrenal tumor of pulmonary adenocarcinoma

We report a case of pulmonary adenocarcinoma metastasizing to the adrenal glands, which caused adrenal insufficiency leading to impaired consciousness. A 62 year-old man was admitted with impaired consciousness. The patient started chemotherapy from 2004 for pulmonary adenocarcinoma. In August 2004, a metastatic adrenal tumor was detected and chemotherapy was continued thereafter. From July 2005, the patient started to have mild hyperkalemia, anorexia and general malaise, which progressed to disturbance of consciousness. At admission, physical examination showed generalized pigmentation in the skin and mucosa. Blood test revealed hypoglycemia, hyponatremia and hyperkalemia. A dexamethasone suppression test and a rapid ACTH loading test led to a diagnosis of primary hypoadrenalism (Addison's disease). Treatment with hydrocortisone improved the physical status and blood test values. However, the patient subsequently died of disseminated intravascular coagulation due to the tumor.     

Altered Ribosomes in Antibiotic-Resistant Mutants of Mycobacterium smegmatis

Two alleles for viomycin-capreomycin resistance (vic) in Mycobacterium smegmatis affect ribosome structures. One (vicA) affects a component of 50S subunits and the other (vicB) affects a component of 30S subunits. The locus for neomycin-kanamycin resistance (nek), which is linked to vicA and vicB, affects a component of 30S subunits. Although the erythromycin resistance locus (ery) is linked to vic and nek, no ribosomal alterations could be detected. Mutations at the streptomycin locus (str) not linked to vic and nek caused alterations of 30S subunits.     

Antibiotic susceptibility of <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> strains isolated from various clinical sources in Hokkaido Prefecture,Japan

Any increase in β-lactam-resistant Haemophilus influenzae is a serious problem in respiratory and otolaryngology medicine. In this study, we examined the antibiotic susceptibility and genotype of 457 clinical Haemophilus influenzae strains isolated in Hokkaido Prefecture, Japan. Strains with β-lactam-resistant mutations in gene encoding penicillin-binding protein 3 were more frequently found in lower respiratory tract specimens (sputa) than in upper respiratory tract specimens, such as rhinorrhea. The existence of the TEM-1 β-lactamase gene occurred more frequently in adult patients than in pediatric patients. The results suggest that β-lactam-resistant or nonsusceptible strains are more prevalent in adult patients with respiratory diseases. We observed only a very few strains which were nonsuscpetible to third-generation cephalosporins (CEPs) and carbapenems. However, 12%–13% of the strains were shown to be resistant to penicillins and second-generation CEPs, and approximately 4% of the strains were shown to be nonsusceptible to fourth-generation CEPs. In addition, we identified tetracycline-resistant (2.8%), chloramphenicol-resistant (0.6%), clarithromycin-resistant (2.6%), and fluoroquinolone-nonsusceptible (approximately 2%) H. influenzae strains. Y. Ohkoshi and S. Yokota contributed equally to this work.     

MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland

Malarial sporozoites mature in the oocysts formed in the mosquito midgut wall and then selectively invade the salivary glands, where they wait to be transmitted to the vertebrate host via mosquito bite. Invasion into the salivary gland has been thought to be mediated by specific ligand-receptor interactions, but the molecules involved in these interactions remain unknown. MAEBL is a single transmembrane-like protein that is structurally related to merozoite adhesive proteins. We found MAEBL of the rodent malaria parasite, Plasmodium berghei, to be specifically produced by the sporozoites in the oocyst and localized in their micronemes, which are secretory organelles involved in malarial parasite invasion into the host cell. A targeted disruption experiment of the P. berghei MAEBL gene revealed that it was essential for sporozoite infection of the salivary gland and was involved in the attachment to the salivary gland surface. In contrast, the disruption of the MAEBL gene did not affect sporozoite motility in vitro nor infectivity to the vertebrate host. These results suggest that P. berghei MAEBL is a sporozoite attachment protein that participates in specific binding to and infection of the mosquito salivary gland.     

Abnormal cardiac [123I]‐meta‐iodobenzylguanidine uptake in multiple system atrophy

[¹²³I]‐Meta‐iodobenzylguanidine (MIBG) myocardial scintigraphy is useful for distinguishing multiple system atrophy (MSA) from Parkinson disease. In this study, longitudinal observation using MIBG myocardial scintigraphy was carried out in patients with MSA to evaluate the association of myocardial MIBG uptake with clinical features. A total of 96 MIBG examinations were performed in 52 patients with MSA. The heart/mediastinum (H/M) ratio of MIBG uptake at 240 minutes after injection was below the lower limit in 16 patients with MSA (31.3%). Overall, the H/M ratio correlated with neither disease duration nor severity. In the follow‐up observations, the H/M ratio did not show any specific trends, in contrast with the continuous decrease observed in patients with Parkinson's disease. This data clearly showed that cardiac MIBG uptake cannot necessarily be preserved in patients with MSA and that approximately 30% of patients with MSA showed decreased MIBG uptake without any correlation to disease duration or severity. © 2010 Movement Disorder Society