Mn3Ge-based tetragonal Heusler alloy thin films with addition of Ni, Pt, and Pd.

We have investigated substitution effects of Ni, Pt, and Pd on phase formation and magnetic properties of D022-Mn3Ge thin films. We prepared (Mn1-x M x )3Ge thin films (M = Ni, Pt, Pd) at 650 °C by magnetron sputtering on MgO(0 0 1) substrates with x varying from 0.03 to 0.6. For improving the film quality, a Cr(0 0 1) seed layer was employed. The D022 structure formed only for the lowest concentrations of Ni and Pt. Nevertheless, the doped samples showed strong perpendicular magnetic anisotropy up to x = 0.1. For high Ni concentrations, we observed the formation of a soft ferromagnetic Mn x Ni y Ge phase with a Curie temperature of about 230 K, while in samples with high Pt content the antiferromagnet L10-MnPt phase is formed along with GePt. In contrast, for Pd substitution, the D022 structure is preserved up to x = 0.2, exhibiting strong perpendicular magnetic anisotropy and low saturation magnetization. Interestingly, the coexistence of the D022-Mn3Ge and a novel D022-(Mn1-x Pd x )3Ge phase was revealed, which might have been facilitated by the low lattice mismatch to the Cr(0 0 1) seed layer. With further increase of the Pd concentration, the D022 structure vanishes and mainly the GePd and GePd2 phases are present. Overall within the investigated sample series, the saturation magnetization strongly decreases with increasing dopant concentration, offering the possibility to adjust the saturation magnetization in the range between 20 and 100 emu cm-3, while still preserving strong perpendicular magnetic anisotropy, which is important for spintronic applications.

The common antitussive agent dextromethorphan protects against hyperoxia-induced cell death in established in vivo and in vitro models of neonatal brain injury.

Preterm infants are prematurely subjected to relatively high oxygen concentrations, even when supplemental oxygen is not administered. There is increasing evidence to show that an excess of oxygen is toxic to the developing brain. Dextromethorphan (DM), a frequently used antitussive agent with pleiotropic mechanisms of action, has been shown to be neuroprotective in various models of central nervous system pathology. Due to its numerous beneficial properties, it might also be able to counteract detrimental effects of a neonatal oxygen insult. The aim of the current study was to evaluate its therapeutic potential in established cell culture and rodent models of hyperoxia-induced neonatal brain injury. For in vitro studies pre- and immature oligodendroglial (OLN-93) cells were subjected to hyperoxic conditions for 48 h after pre-treatment with increasing doses of DM. For in vivo studies 6-day-old Wistar rat pups received a single intraperitoneal injection of DM in two different dosages prior to being exposed to hyperoxia for 24h. Cell viability and caspase-3 activation were assessed as outcome parameters at the end of exposure. DM significantly increased cell viability in immature oligodendroglial cells subjected to hyperoxia. In pre-oligodendroglial cells cell viability was not significantly affected by DM treatment. In vivo caspase-3 activation induced by hyperoxic exposure was significantly lower after administration of DM in gray and white matter areas. In control animals kept under normoxic conditions DM did not significantly influence caspase-3-dependent apoptosis. The present results indicate that DM is a promising and safe treatment strategy for neonatal hyperoxia-induced brain injury that merits further investigation.

Maternal testosterone levels are associated with C-peptide levels in the Mexican American subset of the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study cohort.

Altered sex hormone levels are thought to play an important role in adult-onset diseases including obesity, cardiovascular disease, and diabetes. They contribute to these complex diseases through changes in their availability, which is influenced, in part, by binding proteins. Insulin resistance, which is characteristic of these diseases, along with increased insulin secretion, is a physiologic change that occurs normally during pregnancy. To determine the relationship between insulin resistance and sex hormone levels, we examined the associations of sex hormone-binding globulin (SHBG) and testosterone with measures of glycemia and insulinemia in a healthy pregnant population. We measured fasting serum SHBG and testosterone levels in 215 Hispanic mothers of Mexican ancestry from the HAPO Study cohort and tested for associations between SHBG and testosterone levels and maternal plasma glucose and C-peptide. After adjusting for confounding variables, serum total testosterone (TT) was positively associated with fasting C-peptide (0.18 µg/l higher for TT higher by 1 SD, p=0.001) and 1-h C-peptide (0.79 µg/l higher for TT higher by 1 SD, p<0.001). Free testosterone (FT) was also positively associated with fasting C-peptide (0.19 µg/l higher for FT higher by 1 SD, p<0.001), and 1-h C-peptide (0.83 µg/l higher for FT higher by 1 SD, p<0.001). Although these findings are from a single cohort, this study provides evidence for an association between testosterone and C-peptide during pregnancy in a nondiabetic Hispanic obstetric population.

Dynamic switching of the spin circulation in tapered magnetic nanodisks.

Magnetic vortices are characterized by the sense of in-plane magnetization circulation and by the polarity of the vortex core. With each having two possible states, there are four possible stable magnetization configurations that can be utilized for a multibit memory cell. Dynamic control of vortex core polarity has been demonstrated using both alternating and pulsed magnetic fields and currents. Here, we show controlled dynamic switching of spin circulation in vortices using nanosecond field pulses by imaging the process with full-field soft X-ray transmission microscopy. The dynamic reversal process is controlled by far-from-equilibrium gyrotropic precession of the vortex core, and the reversal is achieved at significantly reduced field amplitudes when compared with static switching. We further show that both the field pulse amplitude and duration required for efficient circulation reversal can be controlled by appropriate selection of the disk geometry.

Neuroprotective effects of the sigma-1 receptor ligand PRE-084 against excitotoxic perinatal brain injury in newborn mice.

Excessive glutamate release followed by N-methyl-D-aspartate receptor (NMDAR) activation plays a crucial role in perinatal brain injury. We have previously shown that dextromethorphan, a low-affinity NMDAR antagonist with anti-inflammatory properties, is neuroprotective against neonatal excitotoxic brain injury. Of interest, dextromethorphan is also a sigma-1 receptor (σ1R) agonist. The pharmacologic class of σ1R agonists has yielded propitious results in various animal models of adult central nervous system pathology. In an established neonatal mouse model of excitotoxic brain injury, we evaluated the effect of the selective σ1R agonist 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE-084). A single intraperitoneal injection of 0.1 µg/g (low dose) or 10 µg/g (high dose) bodyweight (bw) PRE-084, given 1h after the excitotoxic insult, significantly reduced lesion size in cortical gray matter 24 h and 120 h after the insult. Repetitive injections of 0.1 µg/g PRE-084 proved to be equally effective. PRE-084 treatment resulted in a decrease in cell death indicated by reduced TUNEL positivity and caspase-3 activation. Furthermore, it lowered the number of isolectin B4-positive, activated microglial cells. PRE-084 had no effect on developmental apoptosis in the undamaged brain. In vitro findings in primary hippocampal neurons suggest that PRE-084 treatment provides partial protection against glutamate induced morphological and functional changes. For excitotoxicity as playing a crucial role in the pathogenesis of perinatal brain injury, we demonstrate for the first time that systemic treatment with the highly selective σ1R agonist PRE-084 protects against NMDAR-mediated excitotoxic brain damage.

Paraoxonase-1 (PON1) status in pancreatic cancer: relation to clinical parameters.

Human paraoxonase 1 (PON1) has been shown to decrease the level of systemic oxidative stress, which is thought to contribute to cancer development. The aim of this study was to examine the interrelationships between PON1 status and some clinical characteristics in patients with pancreatic cancer (PC). A group of 73 consecutive patients with PC (stage II-IV) and 73 control subjects were examined. Laboratory studies included five polymorphisms of the PON1 gene (L55M, Q192R, -108C/T, -126C/T, and -162A/G), PON1 arylesterase (PON1-A) and lactonase (PON1-L) activities, as well as some markers of protein metabolism, insulin resistance, and oxidative stress. In comparison with the control group, no difference in the distribution of the PON1 polymorphisms was found in cancer patients, both arylesterase and lactonase activities being significantly lower (-33, -47 %, respectively, both P < 0.001). There was neither statistically significant association of PON1 polymorphisms with tumour stages nor with diabetes mellitus connected with PC. The genotype distribution of L55M and ­108C/T differed only in a subgroup of patients presenting clinically relevant malnutrition (χ² = 6.50, 6.25, respectively, both P < 0.05). In the PC group, PON1-A and PON1-L activities correlated with Nutritional Risk Index (r = 0.351, 0.409, respectively, both P < 0.01), PON1-L with mid-arm muscle circumference (r = 0.328, P < 0.05), and PON1-A and PON1-L with serum albumin (r = 0.352, 0.391 respectively, both < 0.01). Our results suggest that PON1 plays an important role in PC, especially in cancer-associated malnutrition.

First assessment of menstrual cycle function and reproductive endocrine status in Samoan women.

BACKGROUND: American Samoa and Samoa are now characterized by one of the world's highest levels of adult overweight and obesity. Our objective was to investigate patterns of menstrual cyclicity reported by Samoan women and examine the relationship to adiposity and select hormone levels. METHODS: A cross-sectional analysis was performed among Samoan women, aged 18-39 years (n = 322), using anthropometric and biomarker measures of adiposity and reproductive health, including insulin, adiponectin, testosterone, sex hormone-binding globulin, free androgen index (FAI) and mullerian-inhibiting substance (MIS). Menstrual regularity was assessed from self-reported responses. Multivariable models were estimated to adjust for potential confounding of the associations between menstrual patterns and other measures. RESULTS: A high proportion of the women (13.7%) reported oligomenorrhea or amenorrhea (OM/AM). More than three-quarters, 80.7%, of women were either overweight or obese, using Polynesian-specific criteria, and OM/AM was significantly associated with higher BMI. Abdominal circumference and insulin levels were significantly higher, and adiponectin levels were lower, in those who reported OM/AM versus regular menstruation. The FAI was higher in women with increased BMI. MIS levels declined with age, more slowly in those reporting OM/AM. CONCLUSIONS: Self-reported OM/AM was associated with an elevated BMI, abdominal adiposity and serum insulin, and with reduced adiponectin levels. These findings support a high rate of metabolic syndrome, and perhaps PCOS and reproductive dysfunction, among Samoan women.

Systemic G-CSF treatment does not improve long-term outcomes after neonatal hypoxic-ischaemic brain injury.

Hypoxia-ischaemia (HI) is a major factor in the pathogenesis of developmental brain injury, leading to cognitive deficits and motor disabilities in preterm infants. The haematopoietic growth factor granulocyte colony-stimulating factor (G-CSF) has been shown to exert a neuroprotective activity in rodent models of ischaemic stroke and is currently subject to phase I/II clinical trials in adults. Results of studies examining the effect of G-CSF in perinatal brain damage have been contradictory. We have previously shown that G-CSF increases NMDAR-mediated excitotoxic brain injury in the neonatal mouse brain. In this study, we evaluated the effect of G-CSF on long-term outcomes after HI. On postnatal day 5, mice pubs were first randomly assigned to a sham operation or HI and then divided into four treatment groups: i) G-CSF; ii) phosphate buffered saline (PBS) 1h after injury; iii) G-CSF and iv) PBS 60 h after injury. G-CSF (200 µg/kg BW) was administered five times within a 24h interval. Neuromotor and cognitive outcomes were assessed by open-field, novel object recognition tests and rotarod tests starting on P90, with subsequent histological analyses of brain injury. G-CSF treatment did not improve either neurobehavioural outcomes or brain injuries. Interestingly, the application of PBS and G-CSF in the acute phase increased brain damage in the hippocampus. We could not confirm the neuroprotective properties of G-CSF in neonatal HI brain damage. The exacerbation of injury by the administration of substances in the acute phase might indicate a heightened state of neurological sensitivity that is specific to mechanisms of secondary neurodegeneration and influenced by unidentified external factors possibly associated with the treatment protocol during the acute phase. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

The role of the polycystic ovary syndrome susceptibility locus D19S884 allele 8 in maternal glycemia and fetal size.

CONTEXT: The high incidence of insulin resistance, type 2 diabetes, and metabolic syndrome in Western societies and their impact on quality of life emphasize the importance of identifying underlying susceptibility loci for metabolic diseases. The polycystic ovary syndrome (PCOS) susceptibility locus D19S884 allele 8 (A8) is associated with measures of insulin resistance, beta-cell dysfunction, and other metabolic phenotypes in PCOS families. We now investigate the role of D19S884 A8 in pregnancy. OBJECTIVE: Using the multiethnic Hyperglycemia and Adverse Pregnancy Outcome cohort, we assessed the associations of D19S884 A8 with measures of maternal glycemia and fetal size. DESIGN: We tested for association of maternal D19S884 A8 with maternal outcomes (fasting, 1-h, and 2-h plasma glucose, and fasting and 1-h C-peptide from an oral glucose tolerance test) and fetal and maternal D19S884 A8 with fetal outcomes (birth weight, length, head circumference, sum of skin folds, fat mass, cord C-peptide, and 2-h neonatal plasma glucose). SUBJECTS: We analyzed 4424 Caucasian mothers and 3347 offspring of northern European ancestry, 1957 Thai mothers and 2089 offspring from Bangkok, 1208 Afro-Caribbean mothers and 1209 offspring from Barbados, and 774 Hispanic mothers and 762 offspring from Bellflower, California. RESULTS: After adjusting for confounding variables and multiple testing, neither maternal nor fetal D19S884 A8 showed significant evidence for association with any of the outcomes tested. CONCLUSIONS: The PCOS susceptibility locus, D19S884 A8, is not a major factor contributing to glycemia during pregnancy or fetal size in a general obstetric population.

Focused ion beam fabrication of spintronic nanostructures: an optimization of the milling process.

Focused ion beam (FIB) milling has been used to fabricate magnetic nanostructures (wires, squares, discs) from single magnetic layers (Co, permalloy) and spin-valve (permalloy/Cu/Co) multilayers (thicknesses 5-50 nm) prepared by ion beam sputtering deposition. Milled surfaces of metallic thin films typically exhibit residual roughness, which is also transferred onto the edges of the milled patterns. This can lead to domain wall pinning and influence the magnetization behaviour of the nanostructures. We have investigated the milling process and the influence of the FIB parameters (incidence angle, dwell time, overlap and ion beam current) on the roughness of the milled surface. It has been found that the main reasons for increased roughness are different sputter yields for various crystallographic orientations of the grains in polycrystalline magnetic thin films. We have found that the oblique ion beam angle, long dwell time and overlap < 1 are favourable parameters for suppression of this intrinsic roughness. Finally, we have shown how to determine the ion dose necessary to mill through the whole thin film up to the silicon substrate from scanning electron microscopy (SEM) images only.

Gender and neurogenin3 influence the pathogenesis of ketosis-prone diabetes.

Ketosis-prone diabetes (KPD) is a phenotypically defined form of diabetes characterized by male predominance and severe insulin deficiency. Neurogenin3 (NGN3) is a proendocrine gene, which is essential for the fate of pancreatic beta cells. Mice lacking ngn3 develop early insulin-deficient diabetes. Thus, we hypothesized that gender and variants in NGN3 could predispose to KPD. We have studied clinical and metabolic parameters according to gender in patients with KPD (n = 152) and common type 2 diabetes (T2DM) (n = 167). We have sequenced NGN3 in KPD patients and screened gene variants in T2DM and controls (n = 232). In KPD, male gender was associated with a more pronounced decrease in beta-cell insulin secretory reserve, assessed by fasting C-peptide [mean (ng/ml) +/- s.d., M: 1.1 +/- 0.6, F: 1.5 +/- 0.9; p = 0.02] and glucagon-stimulated C-peptide [mean (ng/ml) +/- s.d., M: 2.2 +/- 1.1, F: 3.1 +/- 1.7; p = 0.03]. The rare affected females were in an anovulatory state. We found two new variants in the promoter [-3812T/C (af: 2%) and -3642T/C (af: 1%)], two new coding variants [S171T (af: 1%) and A185S (af: 1%)] and the variant already described [S199F (af: 69%)]. These variants were not associated with diabetes. Clinical investigation revealed an association between 199F and hyperglycaemia assessed by glycated haemoglobin [HbA1c (%, +/-s.d.) S199: 12.6 +/- 1.6, S199F: 12.4 +/- 1.4 and 199F: 14.1 +/- 2.2; p = 0.01]. In vitro, the P171T, A185S and S199F variants did not reveal major functional alteration in the activation of NGN3 target genes. In conclusion, male gender, anovulatory state in females and NGN3 variations may influence the pathogenesis of KPD in West Africans. This has therapeutic implications for potential tailored pharmacological intervention in this population.

Fine mapping of genetic susceptibility to polycystic ovary syndrome on chromosome 19p13.2 and tests for regulatory activity.

CONTEXT: Little is known about genes that contribute to polycystic ovary syndrome (PCOS). We previously found linkage and association of PCOS with the dinucleotide marker D19S884 in two independent sets of families; allele 8 of D19S884 confers increased risk. OBJECTIVE/DESIGN: The objectives of the study were: 1) use the transmission/disequilibrium test (TDT) to assess linkage and association between PCOS and D19S884 (and nearby markers) in a third set of families; and 2) test D19S884 and surrounding DNA sequence for in vitro regulatory activity in lymphoblastoid cell lines (LCLs) and granulosa cells. SETTING/SUBJECTS: We studied 98 new families with a PCOS proband, father, mother, and other available offspring. We analyzed data from these families separately and in combination with data obtained previously. INTERVENTIONS: Interventions were venipuncture. MAIN OUTCOME MEASURES: Measures were transmission frequencies and in vitro functional studies. RESULTS: The first result we found was that in the 98 new families, the TDT was significant for allele 8 of D19S884 (P = 0.043). In the total collection of 465 families, the TDT evidence is very strong (nominal P < 7 x 10(-5)). Results for all other genetic markers near D19S884 were nonsignificant after correction for multiple testing. The second result was that an approximately 800-bp fragment containing various alleles of D19S884 showed modest but reproducible promoter activity in LCLs. However, no allelic differences were detected. No activity of this fragment was detected in granulosa cells. CONCLUSIONS: This is the second independent confirmation of linkage and association of D19S884 with PCOS. We found in addition that some sequence in the region of D19S884 confers in vitro promoter activity in LCLs.

Candidate gene region for polycystic ovary syndrome on chromosome 19p13.2.

CONTEXT: Polycystic ovary syndrome (PCOS) is a common endocrine disorder that is believed to have a genetic basis. However, no specific susceptibility gene or region has been conclusively identified. OBJECTIVE: The objective of this study was to duplicate a previous study that localized a PCOS susceptibility region to chromosome 19p13.2 and to narrow the susceptibility region. DESIGN: This study was designed to test for genetic linkage and association between PCOS and short tandem repeat polymorphisms in 367 families, by analysis of linkage and family-based association. SETTING: The study was conducted at academic medical centers. PATIENTS OR OTHER PARTICIPANTS: We studied 367 families of predominantly European origin with at least one PCOS patient. Families included 107 affected sibling (sister) pairs (ASPs) in 83 families, and 390 trios with both parents and an affected daughter. The data set comprises two independent groups. Set 1 consists of 44 ASPs and 163 trios. Set 2 consists of 63 ASPs and 227 trios. INTERVENTION(S): The intervention was the drawing of blood for DNA extraction. MAIN OUTCOME MEASURE: We employed measures of evidence for linkage and association between PCOS and 19 STRs. RESULTS: Linkage with PCOS was observed over a broad region of chromosome 19p13.2. The strongest evidence for association was observed with D19S884 (chi2 = 11.85; nominal P < 0.0006; permutation P = 0.034) and duplicated our earlier findings. CONCLUSIONS: The present analysis suggests that a PCOS susceptibility locus maps very close to D19S884. Additional studies that systematically characterize DNA sequence variation in the immediate area of D19S884 are required to identify the PCOS susceptibility variant.

Determination of bopindolol in pharmaceuticals by capillary isotachophoresis.

Capillary isotachophoresis (ITP) with conductimetric detection has been used for separating and determining bopindolol (I) in commercial mass-produced pharmaceutical preparations. The optimised operational electrolyte system consisted of 5 mM potassium picolinate and 5 mM picolinic acid (leading electrolyte, LE; pH 5.37) and 10 mM formic acid as the terminating electrolyte (TE). The driving and detection currents were 50 microA (for 350 s) and 10 microA, respectively. The single analysis took about 12 min. Under such conditions the effective mobility of I was determined as 16.73 10(-9)m(2) V(-1) s(-1) (with tetraethylammonium as the mobility standard). The calibration graph relating the ITP zone length to the concentration of I was rectilinear (r=0.99990) in the range 10-100 mg l(-1). The relative standard deviation (R.S.D.) was 0.90% (n=6) when determining 50 mg l(-1) of I in pure test solution. Sample pre-treatment of the tablets involved ice-cooled extraction of I with methanol. The method was suitable for determining I in Sandonorm tablets with R.S.D. value 1.45% (n=6). According to the validation procedure based on the standard addition method the recovery was 97.3%.

Searching for the polycystic ovary syndrome genes.

PCOS is a common disorder of unknown etiology. Studies of first-degree relatives of women diagnosed with PCOS suggest familial clustering of the disease. A prospective study of first-degree female relatives of women with PCOS conducted by NCPIR found that 46% of ascertainable sisters of women with PCOS were hyperandrogenemic. NCPIR has conducted linkage and association studies using affected sibling-pair analysis and the transmission/disequilibrium test to explore candidate PCOS genes. These studies point a finger at a region 1 MB centromeric to the insulin receptor gene on chromosome 19.

Localization of a hereditary neuroblastoma predisposition gene to 16p12-p13.

BACKGROUND: Hereditary predisposition to develop neuroblastoma segregates as an autosomal dominant Mendelian trait. PROCEDURE: We have performed linkage analysis on 10 families with neuroblastoma to localize a hereditary neuroblastoma predisposition gene (HNB1). RESULTS: A single genomic interval at chromosome bands 16p12-p13 was consistent with linkage (lod = 3.46), and identification of informative recombinants defined a 25.9-cM critical region between D16S748 and D16S3068. Loss of heterozygosity was identified in 5/12 familial (42%) and 55/259 nonfamilial (21%) neuroblastomas at multiple 16p polymorphic loci. A 12.8-cM smallest region of overlap of deletions was identified within the interval defined by linkage analysis (tel-D16S764-D16S412-cen). CONCLUSIONS: Taken together, these data suggest that HNB1 is located at 16p12-p13 and that inactivation of this gene may contribute to the pathogenesis of nonfamilial neuroblastomas.

Reduction of tumor burden and stabilization of disease by systemic therapy with anti-CD20 antibody (rituximab) in patients with primary cutaneous B-cell lymphoma.

BACKGROUND: Primary cutaneous B-cell lymphoma (CBCL) is characterized by restriction to the skin, a high incidence of recurrence after various treatment modalities, and a variable but mostly favorable prognosis. METHODS: Ten patients with long standing primary CBCL (3 with follicular CBCL, 5 with cutaneous, large B-cell lymphoma, 1 with diffuse large cell lymphoma, and 1 with extranodal large cell lymphoma) were treated by intravenous application of a chimeric antibody against the CD20 transmembrane antigen that is present on malignant and normal B-cells. In 6 of 10 patients, several treatment attempts either had failed or could not be used due to severe side effects or underlying disease. RESULTS: The treatment regimen resulted in two complete regressions, five partial responses, and one mixed response, and two patients did not respond to the treatment. No severe side effects occurred, except for slight pain in the nodules after infusion and an urticarial reaction at the tumor sites. A prolonged, complete disappearance of B-cells from the peripheral blood was observed. The immunoglobulin serum levels and inflammatory markers were unchanged. Histologic examination of biopsies from two regressing tumor nodes showed necrotic tumor cells and infiltration with CD8 positive cells. CONCLUSIONS: Intravenous therapy with the anti-CD20 antibody rituximab is a nontoxic and effective treatment for patients with primary cutaneous B-cell lymphoma.

Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19.

Various molecular markers have been used for the detection of circulating breast cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). Using nested RT-PCR, we compared the specificity and sensitivity of human mammaglobin (hMAM), epidermal-growth-factor receptor (EGF-R), and cytokeratin 19 (CK-19) expression as markers for circulating carcinoma cells in the peripheral blood of patients with breast cancer. Blood samples from 12 patients with ductal carcinoma in situ, 133 patients with invasive breast cancer, 20 patients with hematological malignancies, 31 healthy volunteers, and tumor tissues from 40 patients with invasive breast cancer were screened for mRNA encoding hMAM, EGF-R, or CK-19 by nested RT-PCR. In all breast cancer tissues, mRNA for hMAM, EGF-R, and CK-19 was detectable. In blood samples from patients with invasive breast cancer, 11 (8%), 13 (10%), and 64 (48%) were positive for mRNA encoding hMAM, EGF-R, or CK-19, respectively. Blood samples from none of the healthy volunteers and patients with hematological disorders were positive for hMAM, while CK-19 mRNA was found in the blood of 12 (39%) healthy volunteers and transcripts for EGF-R and CK-19 were detectable in 5 (25%) and 2 (10%), respectively, of the patients with hematological malignancies. Only hMAM mRNA expression in blood correlated with clinical parameters such as nodal status, metastasis, and CA 15-3 serum levels. In summary, hMAM transcripts detectable in blood by RT-PCR represent the most specific molecular marker for hematogenous spread of breast cancer cells. With the nested RT-PCR method, aberrant EGF-R mRNA expression might occasionally be found in hematological malignancies, whereas CK-19 mRNA expression proved to be rather nonspecific. The prognostic value of hMAM RT-PCR-based tumor cell detection in peripheral blood should be further tested and validated in prospective studies.

Capillary isotachophoretic determination of flufenamic, mefenamic, niflumic and tolfenamic acid in pharmaceuticals.

Anionic capillary isotachophoresis (ITP) with conductimetric detection has been used for determining selected non-steroid anti-inflammatory and analgesic drugs of the phenamate group, namely tolfenamic (I), flufenamic (II), mefenamic (III) and niflumic (IV) acid. Initially the pKa values (proton lost) of I-IV were determined as 5.11, 4.91, 5.39 and 4.31, respectively, by the UV spectrophotometry in aqueous 50% (w/w) methanol. The optimised ITP electrolyte system consisted of 10 mM HCl + 20 mM imidazole (pH 7.1) as the leading electrolyte and 10 mM 5,5'-diethylbarbituric acid (pH 7.5) as the terminating electrolyte. The driving and detection currents were 100 microA (for 450 s) and 30 microA, respectively (a single analysis took about 20 min). Under such conditions the effective mobilities of I-IV varied between 23.6 and 24.6 m2 V(-1) s(-1) (evaluated with orotic acid as the mobility standard). The calibration graphs relating the ITP zone length to the concentration of the analytes were rectilinear (r = 0.9987-0.9999) in the range 10-100 mg l(-1) of the drug standard. The R.S.D.s were 0.96-1.55% (n = 6) when determining 50 mg l(-1) of the analytes in pure test solutions. The method has been applied to the assay of the phenamates in six commercial mass-produced pharmaceutical preparations (Mobilisin gel and ointment, Lysalgo capsules, Nifluril cream, Niflugel gel, and Clotam capsules). According to the validation procedure based on the standard addition technique the recoveries were 98.4-104.3% of the drug and the R.S.D. values were 1.25-3.32% (n = 6).