Familial hypocalciuric hypercalcemia (FHH) type 1, caused by a heterozygous inactivating mutation of the gene encoding the calcium-sensing receptor (CaSR), is characterized by mild to moderate ...hypercalcemia, hypocalciuria and inappropriately normal or elevated parathyroid hormone (PTH). FHH must be differentiated from primary hyperparathyroidism (PHPT) because parathyroidectomy is ineffective in the former. Herein, we report a 39-year-old male patient with a 13-year history of asymptomatic PTH-dependent hypercalcemia (mean calcium of 2.88 mmol/l; reference range 2.15-2.55 mmol/l) and calcium-to-creatinine clearance ratio (Ca/Cr) ranging from 0.007 to 0.0198, which is consistent with either FHH or PHPT. Although a family history of hypercalcemia was negative, and PET-CT with fluorocholine was suggestive of a parathyroid adenoma, genetic analysis of the CaSR gene identified a heterozygous inactivating mutation NM_000388.4:c.1670G>A p. (Gly557Glu) in exon 6 and a polymorphism NM_000388.4:c.1192G>A p. (Asp398Asn) in exon 4. The G557E mutation has been previously reported in a Japanese family in which all family members with the mutation had Ca/Cr below 0.01 consistent with FHH. The biochemical profile of FHH and PHPT may overlap. Our FHH patient with a G557E CaSR mutation illustrates that the differential diagnosis can be difficult in an index case with no family history, (false) positive parathyroid imaging and higher calciuria than expected for FHH. Calcium intake, vitamin D status and bone resorption might have contributed to the Ca/Cr variations over a 13-year clinical follow up. This case thus emphasizes the irreplaceable role of genetic testing of the CaSR gene when clinical evaluation is inconclusive.
Abstract The recent return of samples from asteroid 162173 Ryugu provides a first insight into early Solar System prebiotic evolution from known planetary bodies. Ryugu’s samples are CI ...chondrite-like, rich in water and organic material, and primarily composed of phyllosilicate. This phyllosilicate surrounds micron to submicron macromolecular organic particles known as insoluble organic matter. Using advanced microscopy techniques on Hayabusa-2 samples, we find that aqueous alteration on Ryugu produced organic particles richer in aromatics compared to less altered carbonaceous chondrites. This challenges the view that aromatic-rich organic matter formed pre-accretion. Additionally, widespread diffuse organic material occurs in phyllosilicate more aliphatic-, carboxylic-rich, and aromatic-poor than the discrete organic particles, likely preserving the soluble organic material. Some organic particles evolved to encapsulate phyllosilicate, indicating that aqueous alteration on Ryugu led to the containment of soluble organic matter within these particles. Earth therefore has been, and continues to be, delivered micron-sized polymeric organic objects containing biologically relevant molecules.
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only ...method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.
An experiment evaluated the influence of several variables on the coumestrol content of alfalfa (
Medicago sativa) silages. Two cultivars of alfalfa (Morava and Palava) were each harvested on May, ...June, and October 2000. The forage was allowed to wilt in the swath to approximately 300 or 500
g/kg dry matter (DM) content and then it was ensiled without further treatment. The number of lactic bacteria, mold, and yeast as well as pH and concentration of lactic acid were indicative of adequate preservation. The majority of the initial content of coumestrol disappeared during 50 days of ensiling, but some was still detected after 150 days. The coumestrol concentration in silages varied with both cultivar and maturity of plant while there was no significant effect of the dry matter content of feed forage. This report shows that wilting in the swath caused remarkable increase in coumestrol content of alfalfa.
The separation of six isoflavones (biochanin A, isoformononetin, formononetin, prunetin, daidzein and genistein) and coumestrol on an uncoated fused-silica capillary electrophoresis column was ...optimised using alkaline borate buffer as electrolyte and DAD detection. A baseline separation of all analytes except a pair, formononetin-biochanin A was achieved at pH 10.5 in 25 min. Detection limits were low (0.1 microg/mL) and the linearity of the detector response was established in the concentration range 0.4-60 microg/mL (180 microg/mL for coumestrol). Coumestrol was synthesized and the carbon signals in 13C-NMR spectrum of both coumestrol and di-O-acetylcoumestrol were assigned for the first time using two-dimensional HMQC technique.
We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. ...Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.
In the prospective study, we examined hematopoietic mixed chimerism (using polymerase chain reaction (PCR) of variable number of tandem repeat-VNTR sequences) and minimal residual disease (MRD) ...status (using qualitative and in the case of positivity quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR/ABL fusion mRNA) in serial peripheral blood samples taken from 25 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Increasing mixed chimerism in correlation with increasing signal of MRD was detected in 10 patients. In two patients mixed chimera status and BCR/ABL rearrangement led to hematologic relapse, in five patients molecular relapse was followed by reappearance of Ph chromosome and three patients developed molecular relapse only. Adoptive immunotherapy-donor lymphocyte infusion (DLI), interferon (INF) and discontinuation of post-transplant immunosupression-separately or in different combinations was used in nine patients with molecular, cytogenetic or hematologic relapse of CML. The results demonstrate that significant response at the molecular level can be achieved for a majority of CML patients and that using of all forms of adoptive immunotherapy controlled by MC and MRD is more efficient in patients treated in early molecular relapse-with minimal disease burdens.
Chinese herbal medicine is gaining increasing popularity worldwide for health promotion and adjuvant therapy. Thus, selective and efficient analytical methods are required not only for quality ...assurance but also for authentication of the plant material. Applications of both chromatographic and electrophoretic techniques to the analysis of medicinal plants and Chinese traditional medicine preparations over the last 3 years are outlined in this review. The role of chemical fingerprinting is highlighted and a brief survey of determination of toxic components, natural and synthetic adulterants is also included. Moreover, different sample pretreatment and extraction methods are discussed.
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