An approach for plasma cell myeloma diagnosis by two-color flow cytometry based on kappa/lambda ratios of CD38-gated plasma cells.

Criteria from the World Health Organization (WHO) are commonly used to diagnose plasma cell myeloma (PCM), but they are complex and require several laboratory parameters. To differentiate reactive plasmacytosis from clonal plasma cell neoplasms, such as PCM, it is important to accurately determine the expression of the cytoplasmic immunoglobulin (cIg) light chain (LC). Through retrospective analyses, we selected the patients with PCM, and analyzed records of 52 PCM patients, who underwent bone biopsies, and final diagnosis of PCM was established according to WHO criteria, and 22 controls. In the present study, all samples were analyzed by flow cytometry (FC) in the side scatter vs CD38 histogram mode, and the CD38-gated plasma cell population was identified. The positive cell ratios of kappa and lambda to plasma cell populations were analyzed. PCM cells were distinguished from normal plasma cells by a cut-off level between 0.80 and 3.3, a sensitivity of 90.3 percent, and a specificity of 81.1 percent. Two-color FC analysis is simple to perform, inexpensive, and clinically relevant data are obtained soon after completion of the FC measurements. It could be one of the helpful tools in the diagnosis of PCM. The correct diagnosis of PCM can be achieved more simply, efficiently, and rapidly by combining this method.

Terminal nerve gonadotrophin-releasing hormone (GnRH) neurones express multiple GnRH receptors in a teleost, the dwarf gourami (Colisa lalia).

Gonadotophin-releasing hormone (GnRH) peptide released from the terminal nerve (TN)-GnRH neurones of the dwarf gourami primarily modifies the electrical properties of various neurones, including the TN-GnRH neurones themselves. However, our knowledge on the expression of GnRH receptors (GnRHRs) in the TN-GnRH neurones is still limited. Here, we used the single-cell reverse transcriptase-polymerase chain reaction after whole-cell patch-clamp recording to study the distribution of various GnRHR types expressed in the individual TN-GnRH neurones. We found that TN-GnRH neurones express two of the three types of GnRHRs cloned in the dwarf gourami: GnRHR1-2 and -R2, but not -R1-1. Furthermore, in agreement with our previous findings, all TN-GnRH neurones contained mRNAs of salmon GnRH but not chicken GnRH-II.

Loss of Secreted Frizzled-Related Protein-1 expression is associated with poor prognosis in intrahepatic cholangiocarcinoma.

AIMS: Secreted Frizzled-Related Protein-1 (SFRP1) is a well-known negative regulator of the wingless type (Wnt)-ß-catenin pathway and its inactivation plays an important role in the development and progression of many cancers. In this study, we aimed to determine the clinical significance of SFRP1 expression in intrahepatic cholangiocarcinoma (IHCC) and to define the relationship to Wnt-ß-catenin pathway. METHODS: Fifty IHCC patients who had liver resection were enrolled in this study. SFRP1 protein expression was examined by immunohistochemistry in tumor tissues. The patients were divided into two groups: SFRP1 positive (n = 30) and negative (n = 20). Clinicopathological characteristics were analyzed. RESULTS: SFRP1 significantly correlated with curability (Cur A, B vs. C, p = 0.029); and recurrent pattern (intrahepatic vs. extrahepatic, p = 0.010). The negative SFRP1 group had significantly poorer prognosis, and 5-year survival rates were 8.1% of the negative SFRP1 group and 44.6% of the positive SFRP1 group, respectively. Moreover, the disease-free survival rate in the negative SFRP1 group was significantly poorer (p < 0.001). Multivariate analysis revealed that loss of SFRP1served as an independent prognostic factor in IHCC for both overall (HR, 2.923; 95% CI, 1.30-6.56; p = 0.009) and disease-free (HR, 2.631; 95% CI, 1.31-5.27; p = 0.006) survival. In addition, SFRP1 expression negatively correlated to ß-catenin expression (p = 0.005). CONCLUSIONS: Those results suggested that the loss of SFRP1 could be a poor prognostic factor for IHCC, through the Wnt-ß-catenin pathway.

Identification and molecular characterization of three GnRH ligands and five GnRH receptors in the spotted green pufferfish.

Gonadotropin-releasing hormone (GnRH) is thought to have diverse physiological functions. Understanding regulatory mechanisms of GnRH functions requires detailed knowledge of gene expressions of both GnRH ligands and receptors in a single species. This report concerns identification and molecular characterization of GnRH ligands and receptors in the spotted green pufferfish Tetraodon nigroviridis. It was identified that the pufferfish possessed three types of GnRH ligands and five types of GnRH receptors. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. Gonads expressed all the ligand and receptor subtypes. Two of five receptor subtypes could not be detected in the pituitary gland of reproductively active individuals, suggesting the existence of novel GnRH systems independent of hypothalamic-pituitary-gonadal axis. Alternative splicing was also observed for some receptor subtypes. The present results indicate that diversified gene expressions combined with molecular diversity contribute to the functional diversity of GnRH.

Evaluation of the Microsemi CRP, an automated hematology analyzer for rapid 3-part WBC differential and CRP using whole blood.

INTRODUCTION: We evaluated the basic performance of Microsemi CRP, an unique automated hematology analyzer which can simultaneously measure CBC including 3-part WBC differential (3-Diff) and CRP using whole blood treated with EDTA-2K anticoagulant. METHOD: We found that it produced generally the acceptable results for all parameters performed (repeatability, reproducibility, linearity, interference effect, carry over, and correlation) using control materials, fresh human whole bloods, and serum samples. RESULTS: CBC data examined using Microsemi CRP showed the good correlation with the previous model, Micros CRP200 (r ≧ 0.9), and also those obtained using the routine analyzer, ADVIA 2120i (r ≧ 0.989). Concerning the 3-Diff, both GRA (%) and LYM (%) showed the excellent correlation coefficient between Microsemi CRP and Micros CRP200 (r ≧ 0.992) as well as ADVIA 2120i (r ≧ 0.957). MON (%) showed good correlation between Microsemi CRP and Micros CRP200 (r = 0.959), but lower correlation between Microsemi CRP and ADVIA 2120 i (r = 0.471). CRP data showed the good correlation with HITACHI7600 (r ≧ 0.997) and Micros CRP200 (r ≧ 0.997). CONCLUSION: From these findings, we concluded that Microsemi CRP seemed the convenient laboratory analyzer in the setting of point of care testing (POCT) especially at NICU or primary care unit.

Eosinophilic peroxidase deficiency: Identification of a point mutation (D648N) and prediction of structural changes.

ABSTRACT Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality without clinical symptoms characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix in eosinophils. Nearly 100 cases have been reported, but a specific mutation has been reported in only one case. We report the genetic analysis of an EPO-deficient subject and his family. The case was found by automated blood analyzer. Sequencing of the entire coding region of the EPO gene disclosed a novel mutation, a 2060 G-A transition (g. 2060G>A) causing an amino acid change from aspartic acid to asparagine (D648N). Both the son and daughter of the propositus inherited the G-A transition, and in vitro expression experiments suggest this transition is responsible for the deficiency. We then analyzed the location of the affected amino acid within this molecule using a structural model of EPO based on myeloperoxidase (MPO). Asn648 is on the inside of the molecule; changing D to N would cause loss of the electrostatic interaction with Arg146 which is crucial for disulfide bonds of the light chain in the N terminus.

Synthesis and pharmacological evaluation of (nitrooxy)alkyl apovincaminates.

A series of (nitrooxy)alkyl apovincaminates has been synthesized and evaluated for their effects on vertebral and femoral blood flow. These derivatives were prepared from apovincaminic acid (4). In cerebral circulation, compound 5 (0.03-1.0 mg/kg iv) caused a dose-dependent increase in cerebral blood flow (CerBF) without affecting the blood pressure. It was more potent than vinpocetine (2). The structures of 2 and 5, determined by X-ray crystallography, showed differences in the electrostatic potential image and in the conformation of the ethyl group at the 16-position.

Properties of Ca(2+) release induced by clofibric acid from the sarcoplasmic reticulum of mouse skeletal muscle fibres.

1. To characterize the effect of clofibric acid (Clof) on the Ca(2+) release mechanism in the sarcoplasmic reticulum (SR) of skeletal muscle, we analysed the properties of Clof-induced Ca(2+) release under various conditions using chemically skinned skeletal muscle fibres of the mouse. 2. Clof (>0.5 mM) released Ca(2+) from the SR under Ca(2+)-free conditions buffered with 10 mM EGTA (pCa >8). 3. Co-application of ryanodine and Clof at pCa >8 but not ryanodine alone reduced the Ca(2+) uptake capacity of the SR. Thus, Ca(2+) release induced by Clof at pCa >8 must be a result of the activation of the ryanodine receptor (RyR). 4. At pCa >8, (i) Clof-induced Ca(2+) release was inhibited by adenosine monophosphate (AMP), (ii) the inhibitory effect of Mg(2+) on the Clof-induced Ca(2+) release was saturated at about 1 mM, and (iii) Clof-induced Ca(2+) release was not inhibited by procaine (10 mM). These results indicate that Clof may activate the RyR-Ca(2+) release channels in a manner different from Ca(2+)-induced Ca(2+) release (CICR). 5. In addition to this unique mode of opening, Clof also enhanced the CICR mode of opening of RyR-Ca(2+) release channels. 6. Apart from CICR, a high concentration of Ca(2+) might also enhance the unique mode of opening by Clof. 7. These results suggest that some features of Ca(2+) release activated by Clof are similar to those of physiological Ca(2+) release (PCR) in living muscle cells and raise the possibility that Clof may be useful in elucidating the mechanism of PCR in skeletal muscle.

Effect of calmodulin antagonists on calmodulin-induced biphasic modulation of Ca(2+)-induced Ca2+ release.

1. Calmodulin (CaM) has a biphasic effect on Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR): potentiation and inhibition at low (pCa > 6.0) and high (pCa 5) Ca2+ concentrations, respectively. To characterize the mode of action of CaM, we studied the effect of CaM antagonists on the CICR in skinned muscle fibres of the rabbit. Ca2+ release was measured by microfluorometry with Fura-2. 2. A CaM antagonist, trifluoperazine (TFP), potentiated the CICR in a dose-dependent manner (10-300 microM) at pCa 6, where a simple reversal of the CaM effect would be inhibition of the CICR. Furthermore, 100 microM TFP sensitized the CICR to Ca2+. A similar effect was produced by other CaM antagonists that were tested: chlorpromazine, W-7, mastoparan, and peptide fragment of CaM-binding residues of CaM-dependent protein kinase II. 3. The biphasic effect of CaM on the CICR was observed even in the presence of high concentrations of CaM antagonists or CaM-bindings peptides. 4. From these results we suggest that CaM has a unique mode of action on the CICR which is quite different from the effect of CaM on known enzymes.

Effects of dantrolene and its derivatives on Ca(2+) release from the sarcoplasmic reticulum of mouse skeletal muscle fibres.

1. We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca(2+) release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle. 2. In intact muscles, GIF-0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF-0082, an azido-functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF-0185. 3. In skinned fibres, Dan inhibited Ca(2+)-induced Ca(2+) release (CICR) under Mg(2+)-free conditions at room temperature. In contrast, GIF-0082 and GIF-0185 showed no inhibitory effect on CICR under the same conditions. 4. Dan-induced inhibition of CICR was not affected by the presence of GIF-0082, whereas it was diminished in the presence of GIF-0185. 5. GIF-0082 and GIF-0185 significantly inhibited clofibric acid (Clof)-induced Ca(2+) release, as did Dan. 6. Several Dan derivatives other than GIF-0082 and GIF-0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof-induced Ca(2+) release. 7. The magnitudes of inhibition of Clof-induced Ca(2+) release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof-induced opening of the RyR-Ca(2+) release channel may be similar to that of physiological Ca(2+) release (PCR). 8. These results indicate that the difference in opening modes of the RyR-Ca(2+) release channel is recognized by certain Dan derivatives.

Identification and characterization of a reptilian GnRH receptor from the leopard gecko.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproductive functions through interactions with its specific receptor. We describe the first molecular cloning and characterization of a full-length GnRH receptor (GnRHR) from the leopard gecko Eublepharis macularius. It has a distinct genomic structure consisting of five exons and four introns, compared with all the other reported GnRHR genes. A native GnRH form, cGnRH-II, stimulated inositol phosphate (IP) production in COS-7 cells transiently transfected with the GnRHR, in a dose dependent manner. The mRNA was expressed in all the tissues and organs examined. Molecular phylogenetic analysis revealed that the cloned GnRHR belongs to the type 2/nonmammalian I GnRHR. Low-expression levels were observed from the pituitary glands of reproductively active leopard geckos, indicating the possibility that there is at least one more type of GnRHR highly expressed in the pituitary gland for the gonadotropin secretion in this reptile.

A novel mutation in the FcgammaRIIIA gene (CD16) results in active natural killer cells lacking CD16.

We report a novel mutation in FcgammaRIIIA (the transmembrane-form CD16) on natural killer (NK) cells in a patient with polyneuropathy. She had no history of recurrent infections. Her NK cells expressed no detectable CD16; however, her NK cytotoxic activity was normal, suggesting that CD16 expression and cytotoxic activity are independent of one another. Molecular analysis revealed a deletion of a single adenine base in exon 4 of CD16 at nucleotide 550. This deletion generates a STOP codon in an extra-cellular domain of the FcgammaRIIIA gene, thereby truncating the CD16 molecule. The patient's NK cells were not recognized by the anti-CD16 monoclonal antibodies 3G8 and Leu11c. Whether the development of her polyneuropathy is associated with this novel mutation is unclear.

Uncoupling mechanism of glycoside antibiotic aculeximycin in isolated rat-liver mitochondria.

Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (Pi)-free medium than in Pi medium. During the uncoupling caused by ACM in Pi medium, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The Pi uptake via Pi/H+ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of Pi/H+ influx through Pi/H+ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates Pi/H+ symporter. However, since the inhibition of Pi/H+ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in Pi-free medium, an increase in the membrane permeability for other ions, such as Na+ and K+, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both Pi and Pi-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.

My4+/LeuM3- molecule and CD19 antigen are down-modulate by low affinity Fc gamma receptor II (CD32) stimulation on CD56-positive B-lymphoma cells.

My4+/LeuM3- molecule is recognized by My4, but not by LeuM3, both well known mAbs to CD14. In a previous study we showed that the My4+/LeuM3- molecule on a human monoblastic cell line, U937, is not CD14, but another cell surface antigen. The roles and functions of the My4+/LeuM3- molecule remained unknown. We now report that specific stimulation of Fc gammaR with aggregated IgG or anti-Fc gammaRII antibody down-modulated the My4+/LeuM3- molecules, as well as CD19, in a case of CD56-positive B cell lymphoma. Stimulation of Fc gammaR with anti-mu antibody, which induced concomitant stimulation of sIg, did not induce down-modulation of either molecule. Stimulation of CR2 (CD21), a protein which is functionally or physically associated with CD19, with anti-CR2 (CD21) mAbs also had no effect. The modulation occurred specifically on CD56-positive B-lymphoma cells, since My4+/LeuM3(-)-positive, CD56-negative B-lymphoma cells did not respond to the stimulation. These results suggest that CD19 and My4+/LeuM3- molecules are functionally or physically associated with Fc gammaR II on CD56 positive B-lymphoma cells defined as being at a terminal B cell differentiation stage.

Effects of androgen receptor polyglutamine tract expansion on proliferation of NG108-15 cells.

Expansion of the polyglutamine tracts in the androgen receptor (AR) has been recognized as a cause of X-linked spinal and bulbar muscular atrophy (SBMA). In the present study, NG108-15 cells were stably transfected with expression vectors coding for either the wild type (WT) AR gene (CAG repeat number = 22) or a mutated (MT) AR gene (CAG repeat number = 52). Cells proliferation and cell cycle parameters were evaluated for NG108-15-WT and NG108-15-MT cells in the presence or absence of androgen. NG108-15-WT cells demonstrated an androgen-dependent increase in cell number, while NG108-15-MT cells did not. Our results demonstrate that expansion of polyglutamine tracts in the AR may affect the proliferation and differentiation of nerve cells.

Actions of the novel oral antidiabetic agent HQL-975 in insulin-resistant non-insulin-dependent diabetes mellitus model animals.

The hypoglycemic effects of a novel oral antidiabetic agent HQL-975, were studied in normal rats, streptozotocin-induced diabetic (STZD) rats and genetically insulin-resistant non-insulin-dependent diabetes mellitus (NIDDM) model animals, KK-Ay mice and Zucker diabetic fatty (ZDF) rats. After the dietary administration of HQL-975 to KK-Ay mice, significant decreases in plasma glucose, insulin, triglyceride and non-esterified fatty acid levels were observed. The effective dosage of HQL-975 to decrease the plasma glucose level by 30% was 3.1 mg/kg per day. However, the plasma glucose level was not altered after the administration of HQL-975 in normal and STZD rats. The results suggest that HQL-975 is more effective against the abnormalities of glucose and lipid metabolism of insulin-resistant model animals than in that of normal and insulin-deficient diabetic animals. It is reported that ZDF rats indicate a severely diabetic state as a result of insulin resistance and further the presence of beta-cell insulin secretory defects. Here, HQL-975 (1-30 mg/kg per day for 7 days) was administered to ZDF rats; slight decreases in the plasma glucose (18%) and lipids (41%) levels were observed in the rats given 30 mg/kg. To clarify the action mechanism of HQL-975, we studied the effects of HQL-975 administration on the insulin action of target tissues in KK-Ay mice. After the dietary administration of HQL-975 (0.001, 0.003, 0.010% for 7 days) to KK-Ay mice, hepatic glycolytic and gluconeogenic key enzyme activities were measured. The glucose 6-phosphatase activity was decreased (20-40%) as compared with control. The results suggest that HQL-975 enhances the insulin action in hepatic enzyme regulation. To investigate the actions of HQL-975 in peripheral tissues such as muscle and adipose, an in vivo glucose uptake study using 3H-2-deoxyglucose was performed in KK-Ay mice treated with HQL-975 (0.010% for 7 days). The 2-deoxyglucose uptake of the basal state was not altered, but the insulin-stimulated 2-deoxyglucose uptake in muscle (41-191%) and adipose (46-88%) tissues was increased by the HQL-975 treatment as compared with control. These results suggest that HQL-975 also enhances the insulin action of peripheral tissues. Based on these findings, HQL-975 is expected to be useful for treatment of insulin-resistant patients with NIDDM.

Clostridial population and the intestinal lesions in chickens infected with Clostridium perfringens and Eimeria necatrix.

Chickens infected with Clostridium perfringens and Eimeria necatrix were examined bacteriologically and pathologically. When chickens were inoculated with 1.0 x 10(8) C. perfringens and/or 2 x 10(4) E. necatrix sporulated oocysts, populations of C. perfringens in the intestinal contents were examined on 3, 5 and 7 days after E. necatrix inoculation. In both groups infected with E. necatrix, the mean clostridial counts were significantly higher than those of uninfected controls. The concurrent infection had no enhancing effects on increasing the clostridial population more than E. necatrix-alone. Mortality of 4-day-old chickens inoculated on 5 consecutive days with C.perfringens after receiving E. necatrix was higher than those of chickens inoculated with the both organisms. However, intestinal lesions of the concurrently infected group were not different from E. necatrix-alone-infected group on 5 and 7 days after the coccidial infection. When chickens received a large dose (1.5 x 10(9)) of C. perfringens after the inoculation with E. necatrix, edema in the duodenum through jejunum were observed early after the bacterial broth inoculation. These results suggest that the concurrent infection with E. necatrix and C. perfringens increases clostridial population in the intestine of the chickens and has synergic effects on mortality and edema in the upper intestine.

Aculeximycin, a new antibiotic from Streptosporangium albidum. II. Isolation, physicochemical and biological properties.

A new larvicidal antibiotic, aculeximycin, was found in the culture broth of an actinomycete identified as Streptosporangium albidum. Aculeximycin was isolated from the culture filtrate by adsorption on a Diaion HP-20 column and successive elution with acidic aqueous acetone. It was extracted from the concentrated active fraction with 1-butanol and subjected to column chromatography on a Sephadex LH-20 column. Aculeximycin exhibited strong larvicidal activity against mosquito larvae as well as antimicrobial activities against Gram-positive and Gram-negative bacteria, yeasts and molds.

Aculeximycin, a new antibiotic from Streptosporangium albidum. I. Taxonomy of producing organism and fermentation.

A soil isolate of actinomycete, strain TI-1, was found to produce a new antibiotic aculeximycin which killed insects as well as inhibited the growth of bacteria, yeasts and molds in vitro. Yellowish gray colonies on agar media, formation of spherical to oval sporangia at the tip of aerial mycelium and the presence of meso-diaminopimelic acid and madurose in the cell wall ascribed this strain to genus Streptosporangium. From its morphological, cultural and physiological characteristics, the strain was determined to be S. albidum. Production of aculeximycin was carried out by conventional submerged culture: the highest antibiotic titer obtained was 1,250 micrograms/ml.

Structural elucidation of aculeximycin. I. Further purification and glycosidic bond cleavage of aculeximycin.

A new insecticidal antibiotic, aculeximycin (ACM), was produced by an actinomycete identified as Streptosporangium albidum. ACM has been successfully isolated from culture filtrate by a combination of Diaion HP-20, Amberlite CG-50, reversed phase silica gel and Sephadex LH-20 chromatographies. It was found that ACM is a basic glycosidic antibiotic with a molecular weight of 1,672 including five monosaccharide units, three double bonds and a hemiketal ring by preliminary spectral analyses. Treatment of ACM with 1,8-diazabicyclo[5,4,0]undecene-7 caused a glycosidic bond cleavage to give aculexitriose, pseudoaglycones I and II.