104 research outputs found
A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples
Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5′-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes
Monoclonal antibody HBME-1 reacts with a minor subset of B cells with villous surface and can be useful in the diagnosis of hairy cell leukemia and other indolent lymphoproliferations of villous B lymphocytes
The Hector Battifora mesothelial epitope-1 (HBME-1) monoclonal antibody has been generated against human mesothelioma cells and recognizes a biochemically unknown membrane epitope. We have accidentally found that the HBME-1 reacts with scattered lymphocytes showing villous surface in hyperplastic lymphoid tissue. To evaluate its reactivity pattern, we have performed a consecutive immunohistochemical study in nonneoplastic bone marrow and lymphoid samples (n = 40), as well as in malignant lymphoproliferations (n = 427), including hairy cell leukemia (HCL) (n = 72), HCL variant (HCL-v) (n = 13), splenic diffuse red pulp small B cell lymphoma (SDRPL) (n = 8), splenic B cell marginal zone lymphoma (SMZL) (n = 59), and splenic B cell lymphoma/leukemia, not further classifiable on bone marrow morphology (SBCL) (n = 37) cases. The staining pattern of HBME-1 was compared to DBA.44. HBME-1+ villous lymphocytes were constantly detected in low number in nonneoplastic lymphoid tissues. With multicolor immunofluorescence staining, HBME-1+ lymphocytes showed a CD20+/CD79a+/IgM+ B cell phenotype. In B cell lymphoproliferations of villous lymphocytes, HBME-1 reactivity was demonstrated in 96 % of HCL, 39 % of HCL-v, 50 % of SDRPL, 12 % of SMZL, and 19 % of SBCL cases. Nodal and extranodal marginal zone lymphoma cases were positive in 12 % of the cases. A small minority (4 %) of the other B cell lymphomas and no T cell lymphoma revealed tumor cell reactivity with HBME-1. In conclusion, our study has established that HBME-1 reacts with a minor subset of B lymphocytes and a small proportion of B cell lymphomas, which has not been described previously. We suggest that HBME-1 can be a useful marker in the diagnosis of HCL and other indolent lymphoproliferations of villous B lymphocytes
The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro
Aim To assess the effect of 17β-estradiol pretreatment on
the function and expression of α2- adrenergic receptors
(ARs) subtypes in late pregnancy in rats.
Methods Sprague-Dawley rats (n = 37) were treated with
17β-estradiol for 4 days starting from the 18th day of pregnancy.
The myometrial expression of the α2-AR subtypes
was determined by real time polymerase chain reaction
and Western blot analysis. In vitro contractions were stimulated
with (-)-noradrenaline, and its effect was modified
with the selective antagonists BRL 44408 (α2A), ARC 239
(α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate
(cAMP) accumulation was also measured. The
activated G-protein level was investigated by guanosine
5’-O-[gamma-thio]triphosphate (GTPγS) binding assay.
Results 17β-estradiol pretreatment decreased the contractile
effect of (-)-noradrenaline via the α2-ARs, and abolished
the contractile effect via the α2B-ARs. All the α2-AR
subtypes’ mRNA was significantly decreased. 17β-estradiol
pretreatment significantly increased the myometrial cAMP
level in the presence of BRL 44408 (P = 0.001), ARC 239
(P = 0.007), and spiroxatrine (P = 0.045), but did not modify
it in the presence of spiroxatrine + BRL 44408 combination
(P = 0.073). It also inhibited the G-protein-activating effect
of (-)-noradrenaline by 25% in the presence of BRL 44408 +
spiroxatrine combination.
Conclusions The expression of the α2-AR subtypes is sensitive
to 17β-estradiol, which decreases the contractile response
of (-)-noradrenaline via the α2B-AR subtype, and
might cause changes in G-protein signaling pathway. Estrogen
dysregulation may be responsible for preterm labor
or uterine inertia via the α2-AR
Obesity in pregnancy: a novel concept on the roles of adipokines in uterine contractility
Obesity is a global health problem even among pregnant
women. Obesity alters quality of labor, such as preterm labor,
prolonged labor, and higher oxytocin requirements
in pregnant women. The most important factors to play
a role in the altered gestational period and serve as drug
targets to treat the consequences are female sexual hormones,
calcium channels, adrenergic system, oxytocin,
and prostaglandins. However, we have limited information
about the impact of obesity on the pregnant uterine contractility
and gestation time. Adipose tissue, which is the
largest endocrine and paracrine organ, especially in obesity,
is responsible for the production of adipokines and various
cytokines and chemokines, and there are no reliable
data available describing the relation between body mass
index, glucose intolerance, and adipokines during pregnancy.
Recent data suggest that the dysregulation of leptin,
adiponectin, and kisspeptin during pregnancy contributes
to gestational diabetes mellitus and pre-eclampsia. A
preclinical method for obese pregnancy should be developed
to clarify the action of adipokines and assess their impact
in obesity. The deeper understanding of the adipokines-
induced processes in obese pregnancy may be a step
closer to the prevention and therapy of preterm delivery
or prolonged pregnancy. Gestational weight gain is one of
the factors that could influence the prenatal development,
birth weight, and adiposity of newborn
An improved and widely accessible dNTP quantitation tool
Cells maintain a fine-tuned concentration balance in the pool of deoxyribonucleoside 5’-
triphosphates (dNTPs). The perturbation of this balance results in increased mutation
frequencies suggested to promote cancer development and drug resistance. To study dNTP
imbalances and their consequences, an accurate and relatively high-throughput method is
necessary. The dNTP quantitation method of our choice is a fluorescence-based, TaqMan-like
polymerase assay published by Wilson et al, NAR 2011. This assay has the advantages of being
accessible in a standard molecular biology laboratory and having the potential to be automated
in contrast to mass spectrometry or radioactive measurements. Although this method works well
in diluted samples with high dNTP levels, we observed that the sample matrix largely decreases
assay performance.
Upon thorough kinetic analysis of the fluorescent dNTP incorporation curves, we found that
the Taq polymerase exhibits a dNTP independent, signal generating exonuclease activity and
that the polymerization and exonuclease activity are partially inhibited by the sample matrix.
Based on our kinetic investigations we suggest several assay modifications and a novel, kineticsbased
and automated analysis method. Using these modifications, we measured dNTP pools in
widely different organisms including Mycobacterium smegmatis, Staphylococcus aureus and human
cancer cells. We found that our improved method is capable of i) determining dNTP
concentrations in samples previously proved to be unmeasurable by eliminating the interfering
matrix effect, and ii) improving the quantitation limits of the assay.
Fundings: NKFIH-PD 124330, NKFIH-K 115993, János Bolyai Research Scholarshi
High prevalence of Staphylococcus aureus nasal carriage among children in Szolnok, Hungary
We collected nasal samples from 1,390 healthy 3–7 years old children in Szolnok city, Hungary, in 2012. We detected 476 Staphylococcus aureus isolates from 474 children. In two occasions, two different S. aureus were isolated, based on hemolysis type and pulsed-field gel electrophoresis pattern. S. aureus carriage rate was calculated to be 34.1% similar to others studies. Male gender was found to be a risk factor for carriage by statistical analysis. Altogether, four methicillin-resistant S. aureus (MRSA) strains were detected by mecA polymerase chain reaction, which means 0.8% community-acquired MRSA prevalence among the S. aureus isolates. All MRSA strains harbored the SCCmec type IV cassette (typical for CA-MRSA) and belonged to ST45 by multilocus sequence typing. During antibiotic susceptibility testing, we measured the following resistance rates: 0.0% for mupirocin, 0.2% for ciprofloxacin, 0.6% for gentamicin and oxacillin, 3.4% for tetracycline, 9.5% for clindamycin, 10.3% for erythromycin, and 91.4% for penicillin, which are generally lower compared with Hungarian clinical isolates
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