High failure rate of the dissolution tests for 500-mg amoxicillin capsules sold in Cambodia: is it because of the product or the test method?

OBJECTIVES: During the survey of substandard medicines in Cambodia in 2007, it was found that more than 90% of 500-mg amoxicillin (AMPC) capsules failed the United States Pharmacopeia (USP) 30 TEST 1 dissolution test. In the USP, several monographs provide multiple methods for performing the dissolution test. By using the 500-mg AMPC capsule as an example, we aimed to identify the problems and implications of the USP methods adopted for the dissolution test as a global standard. METHODS: All AMPC samples were collected from the Cambodian market in 2007. For the quantitative test, we referred to USP 30. We performed the USP 28 and USP 30 TEST 2 dissolution tests and compared these results with those of the USP 30 TEST 1. RESULTS: All 500-mg AMPC capsules used for the comparison passed the quantitative test. Samples that passed the USP 28 and USP 30 TEST 2 dissolution tests were identical, and the pass rate was 97.1% (34/35), whereas the pass rate with the USP 30 TEST 1 was 8.6% (3/35). The difference in the dissolution results between the three methods was significant (P<0.0001). CONCLUSION: This study revealed that many users would select the most stringent method when multiple methods exist in the USP. This may lead to a high failure rate of the tests. Because USP is a global standard, we recommend that it take into consideration the developing countries and create a more detailed user-friendly manual for selection for appropriate methods.

p21 provides stage specific DNA damage control to preimplantation embryos.

The early stage embryogenesis of higher eukaryotes lacks some of the damage response pathways such as G1/S checkpoint, G2/M checkpoint and apoptosis. We examined here the damage response of preimplantation stage embryos after fertilization with 6 Gy irradiated sperm. Sperm-irradiated embryos developed normally for the first 2.5 days, but started to exhibit a developmental delay at day 3.5. p21 was activated in the delayed embryos, which carried numerous micronuclei owing to delayed chromosome instability. Apoptosis was observed predominantly in the inner cell mass of the day 4.0 embryos. Sperm-irradiated p21-/- embryos lacked the delay, but chromosome instability and apoptosis were more pronounced than the corresponding p21 wild-type embryos. We conclude from the result that damage responses come in a stage-specific manner during preimplantation stage development; p53-dependent S checkpoint at the zygote stage, p21-mediated cell cycle arrest at the morula/blastocyst stages and apoptosis after the blastocyst stage in the inner cell mass.

The supernumerary chromosome of Nectria haematococca that carries pea-pathogenicity-related genes also carries a trait for pea rhizosphere competitiveness.

Fungi are found in a wide range of environments, and the ecological and host diversity of the fungus Nectria haematococca has been shown to be due in part to unique genes on different supernumerary chromosomes. These chromosomes have been called "conditionally dispensable" (CD) since they are not needed for axenic growth but are important for expanding the host range of individual isolates. From a biological perspective, the CD chromosomes can be compared to bacterial plasmids that carry unique genes that can define the habits of these microorganisms. The current study establishes that the N. haematococca PDA1-CD chromosome, which contains the genes for pea pathogenicity (PEP cluster) on pea roots, also carries a gene(s) for the utilization of homoserine, a compound found in large amounts in pea root exudates. Competition studies demonstrate that an isolate that lacks the PEP cluster but carries a portion of the CD chromosome which includes the homoserine utilization (HUT) gene(s) is more competitive in the pea rhizosphere than an isolate without the CD chromosome.

Chromosome complement of the fungal plant pathogen Fusarium graminearum based on genetic and physical mapping and cytological observations.

A genetic map of the filamentous fungus Fusarium graminearum (teleomorph: Gibberella zeae) was constructed to both validate and augment the draft whole-genome sequence assembly of strain PH-1. A mapping population was created from a cross between mutants of the sequenced strain (PH-1, NRRL 31084, originally isolated from Michigan) and a field strain from Minnesota (00-676, NRRL 34097). A total of 111 ascospore progeny were analyzed for segregation at 235 loci. Genetic markers consisted of sequence-tagged sites, primarily detected as dCAPS or CAPS (n = 131) and VNTRs (n = 31), in addition to AFLPs (n = 66) and 7 other markers. While most markers exhibited Mendelian inheritance, segregation distortion was observed for 25 predominantly clustered markers. A linkage map was generated using the Kosambi mapping function, using a LOD threshold value of 3.5. Nine linkage groups were detected, covering 1234 cM and anchoring 99.83% of the draft sequence assembly. The nine linkage groups and the 22 anchored scaffolds from the sequence assembly could be assembled into four chromosomes, leaving only five smaller scaffolds (59,630 bp total) of the nuclear DNA unanchored. A chromosome number of four was confirmed by cytological karyotyping. Further analysis of the genetic map data identified variation in recombination rate in different genomic regions that often spanned several hundred kilobases.

Stimulatory effect of chlorpromazine on prolactin secretion in anencephalic infants.

The effect of chlorpromazine on serum levels of PRL was studied in two anencephalic infants without identifiable hypothalamic tissue. After a single im injection of chlorpromazine, serum concentrations of PRL rose about 3-fold. This result indicates that chlorpromazine stimulates PRL release by directly acting on the pituitary gland in humans.

The effect of gonadotropin-releasing hormone agonist on type I collagen C-telopeptide and N-telopeptide: the predictive value of biochemical markers of bone turnover.

To evaluate the clinical utility of recently developed biochemical markers in the assessment of bone metabolism during GnRH agonist (GnRHa) treatment, we compared five bone resorption markers [C-telopeptide (CTX) and N-telopeptide (NTX) of type I collagen, hydroxyproline (Hpr), pyridinoline (Pyr), and deoxypyridinoline (Dpyr)] and two bone formation markers [total alkaline phosphatase (Alp) and osteocalcin (OC)]. Sixty-eight normally menstruating women were injected with a long-acting GnRHa once a month for 24 weeks for the treatment of endometriosis or leiomyoma. The mean percentage bone loss at the lumbar spine was 3.79% at the end of treatment. Although levels of all markers increased significantly as the treatment progressed, CTX and NTX exhibited the highest correlation coefficients between bone loss at 24 weeks and the seven markers measured at 0, 4, 12, 16, and 24 weeks of treatment. Serum estradiol levels were similarly suppressed during the treatment in both fast losers (whose bone loss was more than the mean) and slow losers (whose bone loss was less than the mean). However, significantly higher z-scores of bone resorption markers, but not of bone formation markers, were observed in the fast losers at 24 weeks of treatment, suggesting a more accelerated bone resorption in this group. Whereas the three highest z-scores at 24 weeks of treatment were CTX, NTX, and Dpyr (in that order), the highest z-score (P < 0.05) was observed for CTX in the fast losers. The subjects in the highest quartile of CTX, the highest, and second highest quartiles of NTX at 24 weeks of treatment experienced 2.1, 2.2, and 1.7 times more bone loss (P < 0.001), respectively, than those in the lowest quartiles. Furthermore, the subjects in the highest quartile of both CTX and NTX experienced 3.6 times more bone loss (P < 0.001) than those in the lowest quartile of both markers. These results indicate that both CTX and NTX are useful and sensitive markers for bone resorption in a hypoestrogenic state induced by GnRHa.

Changes in bone mass as determined by ultrasound and biochemical markers of bone turnover during pregnancy and puerperium: a longitudinal study.

In a longitudinal study, we analyzed the speed of sound (SOS) and broadband ultrasound attenuation (BUA) of the os calcis as an index of bone mineral density (BMD) to define the effects of pregnancy and lactation on bone metabolism. We used an ultrasound bone densitometer and measured 6 biochemical markers of bone turnover in 18 healthy women throughout pregnancy and puerperium. The measurement of SOS and BUA by such an ultrasound device was clinically advantageous; not only is it radiation-free technology, but it also correlates highly with BMD measured by conventional X-ray bone densitometry. While a significant decrease in SOS was found in the 3rd trimester of pregnancy as compared with the early stage of pregnancy, there was no difference in both SOS and BUA between the breast-feeding women and the principally formula-feeding women during a 6-month period of puerperium. The analysis of biochemical markers revealed that both bone formation and bone resorption were elevated in the 3rd trimester of pregnancy as well as during puerperium, and that the breast-feeding women had significantly higher bone metabolism than the principally formula-feeding women. These results indicate that bone mass decreases as bone turnover itself is enhanced during pregnancy, while lactation does not substantially affect bone mass during at least 6 months of puerperium, although bone turnover is active.

Gene expression of epidermal growth factor in human endometrium during decidualization.

Although there are some reports, including our previous study, indicating the existence and biological action of epidermal growth factor (EGF) in human decidua, the site of its synthesis remains unknown. To clarify the EGF production during decidualization at a molecular level, gene expression of EGF in human endometrium/decidua was examined by Northern blot analysis and in situ hybridization. Northern blot hybridization using 32P-labeled human prepro-EGF complementary DNA was carried out for nonpregnant human endometria from hysterectomized uteri of leiomyoma, decidua from early pregnancy, and in vitro decidualized endometrial cells by medroxyprogesterone acetate (MPA). Although no hybridized band was found in the proliferative and secretory phase endometria, a specific band of 5 kilobases, in agreement with the size of human prepro-EGF messenger ribonucleic acid, was detected in decidua of early pregnancy as well as in in vitro MPA-induced decidual cells. In situ hybridization revealed that prepro-EGF messenger ribonucleic acid was observed in the stromal cells of decidua. These results demonstrate that the EGF gene is expressed in the process of decidualization, suggesting that EGF may play an important role in the decidualization process of human endometrium.

Gene expression of gonadotropin-releasing hormone, but not its receptor, in human endometrium and decidua.

Gonadotropin-releasing hormone (GnRH) has been reported to exist in extrahypothalamic tissues such as the placenta, gonads and mammary glands. While we have reported the presence of GnRH-mRNA in the rodent uterus, there have been no reports concerning gene expression of GnRH and its receptor (GnRH-R) in human endometrial tissue. In order to investigate the role of GnRH as a local regulator in the human endometrium, we examined the gene for GnRH and GnRH-R in non-pregnant endometrium and decidua of early pregnancy. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis we found GnRH-mRNA but not GnRH-R-mRNA transcripts in the human endometrium and decidua at 7-9 weeks gestation. This is the first report that suggests GnRH gene expression in the human endometrium/decidua.

Gene expression of double-stranded RNA-dependent protein kinase in the human endometrium and decidua.

To clarify the biological significance of double-stranded RNA-dependent protein kinase (PKR), an interferon (INF)-inducible substance, we investigated (1) PKR gene expression and the (2) effect of IFN-gamma on PKR gene expression in human endometrium. By Northern blot analysis, PKR mRNA was detected as a 2.5 kb band in human endometrium throughout the menstrual cycle and decidua in early pregnancy. The addition of IFN-gamma to culture medium increased the PKR mRNA level in a dose-dependent manner in cultured endometrial stromal cells. These results suggest that IFN-gamma, which is reported to have an inhibitory effect on cell proliferation, plays an important role in human endometrial function by mediating PKR gene expression.

Gene expression and specific binding of platelet-derived growth factor and its effect on DNA synthesis in human decidual cells.

To clarify the biological significance of platelet-derived growth factor (PDGF) in human decidual cell function, which is important for the maintenance of pregnancy, we investigated gene expression of the PDGF subunits, PDGF-A and PDGF-B, specific binding of the PDGF isoform, and the effect of PDGF dimers on DNA synthesis in human decidual cells. We detected in decidua from early pregnancy the expected DNA bands of PDGF-A and PDGF-B by reverse transcriptase-polymerase chain reaction (RT-PCR) as well as mRNAs of each PDGF subunit by Northern blot hybridization, demonstrating that both PDGF subunits exist in this tissue. Scatchard plot analysis showed that decidual cells had both PDGF-alpha and PDGF-beta receptors. PDGF-AA, -AB and -BB stimulated [3H]-thymidine incorporation in cultured decidual cells in a dose-dependent manner. These results indicate the importance of PDGF in human decidua.

Change in C-terminal cross-linking domain of type I collagen in urine, a new marker of bone resorption, during and after gonadotropin-releasing hormone agonist administration.

OBJECTIVE: The major side effect of GnRH agonist (GnRHa) therapy is the reduction of bone mass. To analyze bone resorption by GnRHa, we measured the urinary excretion of C-terminal telopeptides of type I collagen (CTX), a new marker of bone resorption. METHODS: We used a new ELISA for CTX (CrossLaps) in a sample of 18 premenopausal women with leiomyoma who were treated with daily administration of 400 micrograms nafarelin or 900 micrograms buserelin for 16 weeks. RESULTS: Urinary CTX excretion increased significantly during GnRHa treatment and then decreased at 12 and 24 weeks after the cessation of GnRHa therapy. Whereas the excretory profile of CTX during GnRHa therapy was almost similar to that of pyridinoline (Pyr) or deoxypyridinoline (D-Pyr), both biochemical markers of bone resorption, the magnitude of the change in CTX was significantly greater than that in Pyr or D-Pyr. CONCLUSIONS: These results indicate that CTX could be a more sensitive marker for bone resorption than the currently used biochemical markers, and that CrossLaps ELISA is useful for therapeutic monitoring during and after GnRHa treatment.

Effects of epidermal growth factor and transforming growth factor alpha on the mouse trophoblast outgrowth in vitro.

In order to analyze the involvement of growth factors in the implantation mechanism, we examined the direct effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) on trophoblast outgrowth of the mouse blastocyst in vitro. ICR mouse blastocysts were cultured for 4 days on a culture plate in medium containing EGF or TGF-alpha or conditioned medium obtained from cultured endometrial epithelial cells. Blastocysts were also co-cultured with endometrial epithelial cells. The trophoblast outgrowth of these cultured blastocysts was observed daily and the percentage of outgrowing embryos was calculated and analyzed statistically by the chi-squared test. Analysis for the specific binding of 125I-EGF in outgrown trophoblasts was carried out by autoradiography. The co-culture (days 3 and 4) and the presence of EGF (10 ng/ml, day 4), TGF-alpha (1 ng/ml, day 3; 10 ng/ml, days 2 and 3; 50 ng/ml, days 2-4) or conditioned medium (days 3 and 4) significantly stimulated the rate of trophoblast outgrowth. Preincubation of the conditioned medium with monoclonal anti-EGF or anti-TGF-alpha antibody suppressed the stimulatory effect of the conditioned medium on trophoblast outgrowth. The specific 125I-EGF binding in outgrown trophoblasts was demonstrated by autoradiography. These results suggest that EGF and TGF-alpha play an important role in the implantation process by directly stimulating trophoblast development.

Epidermal growth factor stimulates the cell growth of the PA-1 teratocarcinoma cell line in an autocrine/paracrine fashion.

In order to investigate the biological significance of epidermal growth factor (EGF) in the cell function of teratocarcinoma cells, we examined the production, binding and cell proliferative effect of EGF in PA-1 human ovarian teratocarcinoma cell line. The immunoreactivity of EGF in PA-1 cell-conditioned medium was detected by human EGF radioimmunoassay, and prepro-EGF mRNA was demonstrated in PA-1 cells by Northern blot analysis. An [125I]EGF binding study showed the presence of EGF receptor with very high binding affinity and relatively low numbers of binding sites in PA-1 cells. Furthermore, the growth of PA-1 cells was stimulated by EGF and inhibited by anti-EGF monoclonal antibody. These results suggest strongly that EGF plays an important role in controlling the growth of teratocarcinoma cells as an autocrine/paracrine growth factor.

Ovarian strumal carcinoid with severe constipation: immunohistochemical and mRNA analyses of peptide YY.

Functioning ovarian carcinoid tumors are well known to cause carcinoid syndrome. Recently, strumal and trabecular ovarian carcinoid tumors are reported to cause severe constipation possibly because of tumor-producing peptide YY (PYY). We studied a case of primary ovarian strumal carcinoid who had had severe constipation until the tumor was removed by surgical operation. Immunohistochemically, many tumor cells were strongly positive for PYY. By Northern blot and reverse transcription polymerase chain reaction analyses, PYY mRNA was expressed in a complete form as detected in normal human colon mucosa. From these findings, an ovarian strumal carcinoid is strongly suggested to express complete PYY mRNA and therefore complete PYY protein that results in severe constipation.

Cytological Karyotyping of Three Cochliobolus spp. by the Germ Tube Burst Method.

ABSTRACT Cytological karyotypes with mitotic metaphase chromosomes were analyzed for Cochliobolus heterostrophus, C. carbonum, and C. sativus by the germ tube burst method (GTBM). Prior to karyotyping, procedures of GTBM suitable to Cochliobolus were established by examining several crucial conditions such as incubation period of conidia. The estimated chromosome numbers of C. heterostrophus and C. carbonum were n = 15 or 16 and n = 13 or 15 depending on the strains, respectively. In C. sativus, n = 15 was estimated. Morphological information of chromosomes including chromosome size and a threadlike-specific structure representing the nucleolar organizing region was also obtained. Our results for some standard strains are in agreement with previous estimates by pulsed field gel electrophoresis (PFGE) or PFGE coupled with restriction fragment length polymorphism genetic linkage analysis, but inconsistent with the previous estimates for other strains by conventional light microscopic cytology. Additionally, PFGE analysis of C. heterostro-phus strains indicated that chromosome number was not determinable solely by PFGE, which is hampered by comigration and clumping of DNA bands.

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations.

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53 delta, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53 delta cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-ray induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of undifferentiated EC cells differ considerably from those of somatic cells, and that these radioresponses were modulated by a post-irradiation treatment with caffeine.

A solvent extraction-spectrophotometric determination of bismuth(III) as Tetra-n-butylammonium tetraiodobismuthate.

A solvent extraction-spectrophotometric method has been developed for determination of bismuth in the form of tetra-n-butylammonium tetraiodobismuthate(III). The effects of pH, the concentrations of tetra-n-butylammonium and iodide ions, and the nature and amount of the organic, solvent have been studied. Tetra-n-butylammonium was found to be the most useful cation for extraction of tetraiodobismuthate with chloroform, giving quantitative extraction. The optimum pH is <3, and the extracted species is stable for at least a day at room temperature. Bismuth can be determined at the 10(-5)M level in aqueous solution, the relative standard deviation being 1.7%.

Indirect atomic-absorption spectrophotometric determination of phosphorus after flotation as the ion-pair of molybdophosphate with bis[2-(5-chloro-2-pyridylazo)-5-diethylaminophenolato]cobalt(III).

An atomic-absorption spectrophotometric method has been developed for the indirect determination of phosphorus. The calibration graph is linear over the range 0.025-0.2 mug of phosphorus. The relative standard deviation is 1.3% for 0.2 mug of phosphorus (6 replicates). The method has been applied to the determination of phosphate in natural water samples. The enhancement effect of acetone on the determination is discussed.

Conductometric titration of nitrate by formation of the silver-1,10-phenanthroline-nitrate ternary complex.

Nitrate forms an insoluble ternary complex, Ag(phen)(2)NO(3), with silver in the presence of 1,10-phenanthroline (phen). A simple and direct conductometric titration of nitrate, based on the formation of this ternary complex, has been investigated. The sample solution is adjusted to pH 4.3 with acetate buffer and titrated with 0.1M Ag(phen)(+)(2) complex solution in 40% ethanol-water mixture. The relative error is less than 0.5% and relative standard deviation less than 0.4%. The effect of ammonium ion and urea has also been studied. The method has been successfully applied to determination of nitrate-nitrogen in fertilizer.