Measurement of serum transferrin by the γ−γ perturbed angular correlation method

In this work, the average perturbed angular correlation integral attenuation factor G2 of the transferrin in the serums from the normals and the patients with liver cancer were determined using time integral perturbed angular correlation method (PAC). The average values of G2 were 0.1945 +/- 0.0155 for the cancer serum transferrin and 0.2865 +/- 0.0411 for the normal serum transferrin. The difference between these two groups is significant. Mathematical deduction and theoretical analysis suggested that the equivalent radius of the transferrin molecules in the serum of cancer patients becomes smaller than normal, because of a change in the molecular conformation. Similar results were also observed in experiments with rat serum transferrin.     

Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography–coulometric-array detection

The paper describes the computer aided method development and validation for the determination of hydroquinone in human urine from a clinical study on renal excretion of hydroquinone metabolites and the release of free hydroquinone in the urinary tract in order to evaluate the proposed urine disinfecting concept. The presented method uses high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C₁₈, 250×4.6 mm). Selective and sensitive determination (LOQ=12.5 ng on-column) of the target compound was achieved by electrochemical array detection (CoulArray). Gradient and parameter optimization were supported by DryLab software in order to minimize efforts of the expensive and time-consuming method development. Specificity and selectivity were carried out by separation experiments involving the prodrug arbutin and the metabolites hydroquinone, hydroquinone glucuronide, and hydroquinone sulfate, respectively. Hydroquinone glucuronide reference standard was obtained from in vitro glucuronidation in a rat liver microsomes assay. The method was validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998.     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Effect of progesterone pretreatment on the uptake of estradiol-17β by the uterine epithelium of the rat

Summary Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.     

Human MRCKα is regulated by cellular iron levels and interferes with transferrin iron uptake

Myotonic dystrophy kinase-related Cdc42-binding kinase α (MRCKα, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3′-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKα protein expression is also regulated by iron levels; MRCKα colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKα expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKα takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKα activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.     

Structural comparison of mouse and human α-synuclein amyloid fibrils by solid-state NMR

Fibrillar α-synuclein (AS) is the major component of Lewy bodies, the pathological hallmark of Parkinson's disease. Mouse AS (mAS) aggregates much faster than human AS (hAS), although mAS differs from hAS at only seven positions in its primary sequence. Currently, little is known about the site-specific structural differences between mAS and hAS fibrils. Here, we applied state-of-the-art solid-state nuclear magnetic resonance (ssNMR) methods to structurally characterize mAS fibrils. The assignment strategy employed a set of high-resolution 2D and 3D ssNMR spectra recorded on uniformly [(13)C, (15)N], [1-(13)C]glucose, and [2-(13)C]glucose labeled mAS fibrils. An almost complete resonance assignment (96% of backbone amide (15)N and 93% of all (13)C nuclei) was obtained for residues from Gly41 to Val95, which form the core of mAS fibrils. Six β-strands were identified to be within the fibril core of mAS based on a secondary chemical shift and NHHC analysis. Intermolecular (13)C:(15)N labeled restraints obtained from mixed 1:1 (13)C/(15)N-labeled mAS fibrils reveal a parallel, in-register supramolecular β-sheet arrangement. The results were compared in detail to recent structural studies on hAS fibrils and indicate the presence of a structurally conserved motif comprising residues Glu61-Lys80.     

TonB-dependent uptake of β-lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia

The β-lactam antibiotic ceftazidime is one of the handful of drugs with proven clinical efficacy against the important opportunistic human pathogen Stenotrophomonas maltophilia. Here, we show that mutations in the energy transducer TonB, encoded by smlt0009 in S. maltophilia, confer ceftazidime resistance and smlt0009 mutants have reduced uptake of ceftazidime. This breaks the dogma that β-lactams enter Gram-negative bacteria only by passive diffusion through outer membrane porins. We also show that ceftazidime-resistant TonB mutants are cross-resistant to fluoroquinolone antimicrobials and a siderophore-conjugated lactivicin antibiotic designed to target TonB-dependent uptake. This implies that attempts to improve the penetration of antimicrobials into S. maltophilia by conjugating them with TonB substrates will suffer from the fact that β-lactams and fluoroquinolones coselect resistance to these novel and otherwise promising antimicrobials. Finally, we show that smlt0009 mutants already exist among S. maltophilia clinical isolates and have reduced susceptibility to siderophore-conjugated lactivicin, despite the in vitro growth impairment seen in smlt0009 mutants selected in the laboratory.     

β-Galactosidase-deficient human fibroblasts: uptake and processing of the exogenous precursor enzyme expressed by stable transformant COS cells

Summary COS-1 cells were transfected by electroporation with a cDNA for human acid -galactosidase cloned in our laboratory and stable transformants expressing the enzyme activity were selected. The precursor form of the enzyme was secreted in large quantities into the culture medium. The fibroblasts from patients with G_M1-gangliosidosis or Morquio B disease showed a remarkable increase of enzyme activity, up to the normal level, after culture in this medium for 2 days; the amount of uptake was essentially the same as that for the precursor form in human fibroblasts. After endocytosis, the precursor molecules were processed normally to the mature form and remained as stable as those produced by human fibroblasts. On the other hand, cells from galactosialidosis patients did not show any increase of enzyme activity in a similar experiment. It was concluded that the transformants are useful as the source of precursor proteins for the study of intracellular turnover of enzyme molecules in mutant cells.     

Does the regional oxygen uptake measured by near infrared spectroscopy reflect the phase II pulmonary oxygen uptake at the onset of exercise?

It has been hypothesized that the signals of near infrared spectroscopy (NIRS) would reflect muscle O(2) uptake (mVO(2)). Although it is not definite that NIRS signals accurately reflect mVO(2), there is every possibility that NIRS signals at least reflect regional O(2) uptake (rVO(2)). The phase II kinetics of pulmonary oxygen uptake (pVO(2)) is regarded as reflecting mVO(2) at the onset of exercise. To examine whether the rVO(2) on-kinetics measured by NIRS reflects the mVO(2) on-kinetics at the onset of exercise, we compared the rVO(2) as measured by NIRS with the phase II kinetics of pVO(2) at the onset of exercise. Twelve healthy male subjects cycled a Monark ergometer at three different intensities: below the ventilatory threshold (VT) level (below-VT), on the VT level (on-VT), and above the VT level (above-VT), for 6 minutes on three separate occasions. The rVO(2) was calculated from the concentration of oxyhemoglobin and deoxyhemoglobin, as measured by NIRS every 3 seconds. The pVO(2) was determined by the breath-by-breath method. A significant relationship between the amount of increases of pVO(2) and rVO(2) from rest to the end of exercise among all levels of exercise intensity was found (r=0.935, P<0.001). The time constants of rVO(2) (rVO(2)-Tc: below-VT: 6.514+/-2.159 s, on-VT: 7.760+/-2.035 s, above-VT: 9.532+/-2.342 s) were significantly faster than the time constants of pVO(2) (pVO(2)-Tc: below-VT: 23.8+/-4.4 s, on-VT: 25.9+/-5.1 s, above-VT: 26.3+/-5.7 s) (P<0.001). There was no significant relationship between rVO(2)-Tc and pVO(2)-Tc for each intensity (P>0.05). We conclude that the rVO(2) on-kinetics measured by NIRS does not necessarily reflect the mVO(2) kinetics at the onset of exercise.     

Simultaneous determination of eight β-lactam antibiotics in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of eight β-lactam antibiotics, including ampicillin, cefazolin, cefepime, cefmetazole, cefotaxime, doripenem, meropenem, and piperacillin, in human serum. Sample specimens were subjected to solid phase extraction (SPE) using Waters Oasis^® HLB cartridges (30 mg). Chromatographic separation was performed with a high-resolution octadecyl silica column compatible with hydrophilic compounds, using a gradient of 10 mM aqueous ammonium formate containing 0.1% formic acid-methanol. Antibiotics were detected by a triple quadrupole mass spectrometer (MS/MS) with electrospray ionization and quantified by the multiple reaction monitoring mode. A total run time of 13 min was applied. Linearity in the calibration was obtained over a range of 0.1–50 μg/mL of the β-lactam antibiotics, except for doripenem. The lower limit of quantification was 0.005–0.5 μg/mL, using 50 μL serum. The recovery rate exceeded 80.2% for these analytes, except for doripenem (49.1%) and meropenem (62.3%). The present method is applicable to routine therapeutic monitoring of β-lactam antibiotics in clinical practice.     

Effects of succinylacetone on methyl α-d-glucoside uptake by the rat renal tubule

Succinylacetone, a catabolic end-product of tyrosine, is excreted in large quantities in urine from individuals with hereditary tyrosinemia and the Fanconi syndrome. Succinylacetone inhibits rat renal tubular concentrative uptake of the glucose transport analogue, methyl α-d-glucoside, in a noncompetitive and reversible fashion. This compound also depresses oxygen consumption by the rat renal tubule without fine structural damage to mitochondria. It is concluded that succinylacetone may be a useful probe in elucidation of the biochemical mechanism underlying the human Fanconi syndrome.     

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography–mass spectrometry

A GC–MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography–electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-βDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1–20 μg/mL (R² ≥ 0.998). Intra-day accuracies ranged between 97.2–104.9%, 96.1–103.2%, and 97.3–102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2–105.7%, 99.1–105.2%, and 96.5–101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.     

Modulation of formation of the 3’-end of the human argininosuccinate synthetase mRNA by GT-repeat polymorphism

Microsatellites are abundant in the human genome and may acquire context-dependent functions. A highly polymorphic GT microsatellite is located downstream of the poly(A) signal of the human argininosuccinate synthetase (ASS1) gene. The ASS1 participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. To examine possible involvement of the GT microsatellite in ASS1 mRNA 3’-end formation, ASS1 minigene constructs were used in transient transfection for assessment of poly(A) site usage by S1 nuclease mapping. Synthesis of the major human ASS1 mRNA is found to be controlled by two consecutive non-canonical poly(A) signals, UAUAAA and AUUAAA, located 7 nucleotides apart where a U-rich sequence and the GU microsatellite serve as their respective downstream GU/U-rich elements. Moreover, AUUAAA utilization is affected by the GU-repeat number possibly leading to differential regulation of ASS1 polyadenylation in individuals with different repeat numbers. Interestingly, the less efficient UAUAAA motif is noted to be the major ASS1 poly(A) signal possibly as a result of an indispensable downstream U-rich element and restricted utilization of the AUUAAA motif by the presence of extended GU-repeats. The UAUAAA motif and the GT microsatellite are conserved only in primates whereas AUUAAA motif is present in all mammals analyzed. The suboptimal UAUAAA motif and the utilization of the polymorphic GT microsatellite as polyadenylation signal of the ASS1 gene may be used as a strategy in primates to modulate ASS1 level in response to interactions of genetic and environmental factors.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

The control by respiration of the uptake of α-methyl glucoside in Escherichia coli K12

The uptake of methyl α-d-glucopyranoside (α-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells. The inhibition, which occurred only at non-saturating concentrations of α-MG, was not the result of a stimulation of the rate of exit of intracellular α-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide. Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of α-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration.     

Evaluation of uncertainty of measurement from method validation data: An application to the simultaneous determination of retinol and α-tocopherol in human serum by HPLC

An isocratic RP-HPLC method for the determination of retinol and α-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, α-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, α-tocopherol 0.95 mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and α-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; α-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.