Fluorescent Reaction Research Articles

English

Vitamin E is a group of lipid soluble compounds, which are important antioxidants and play a crucial role in mammals and plants. They can protect the membrane from photooxidation, and they are involved in signal transduction and transcription regulation as well. The enzyme homogentisate phytyltransferase (HPT) has been demonstrated to be a key enzyme limiting tocopherol biosynthesis. In this study,HPT gene isolated from Lactuca sativa L. (LsHPT) was transferred into L. sativa(lettuce) via Agrobacterium-mediated method and 15 transgenic plants were obtained. The expression of LsHPT gene and the total content of α and γ-tocopherol in transgenic plants were increased up to 4 and 2.61 folds, respectively, demonstrating that overexpression of HPT gene is an effective way to improve vitamin E content in lettuce.   Key words: Lactuca sativa, tocopherol, homogentisate phytyltransferase (HPT),Agrobacterium-mediated transformation, real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR).

Effect of human cytomegalovirus infection on the expression of Hoxb2 and Hoxb4 genes in the developmental process of cord blood erythroid progenitors

The aim of the present study was to investigate the role of Hoxb2 and Hoxb4 gene expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) on the proliferation and committed differentiation process of human cord blood hematopoietic stem cells (HSCs) to colony-forming erythroid progenitor cells (CFU-Es) in vitro. Cord blood was collected from the fetal placenta umbilical vein in 12 cases and cultured using hematopoietic stem cell culture technique in vitro. The proliferation and differentiation of cord blood HSCs to CFU-Es were continuously disrupted with HCMV-AD169 and/or 6 x 10⁻⁸ mol/l of ATRA. Expression levels of the Hoxb2 and Hoxb4 genes in the blank, ATRA, HCMV-AD169 and ATRA + HCMV treatment groups of CFU-Es were detected on day 3, 7 and 10 of culture by fluorescent quantitative reverse transcriptase-polymerase chain reaction method. Hoxb2 and Hoxb4 gene expression in each group began on day 3, obviously increased on day 7 and reached a peak on day 10. The expression levels of the Hoxb2 and Hoxb4 genes in the HCMV group were obviously down-regulated compared with the level in the blank group. However, expression levels of the Hoxb2 and Hoxb4 genes were significantly up-regulated in the HCMV + ATRA group compared with the HCMV group (P<0.05). Abnormal expression of the Hoxb2 and Hoxb4 genes induced by HCMV may play important roles in abnormal hematopoietic damage. They were also correlated with the process of erythroid hematopoiesis. ATRA (6 x 10⁻⁸ mol/l) significantly up-regulated expression of the Hoxb2 and Hoxb4 genes in the normal erythroid progenitor cells and in those cells infected with HCMV as well.

Spectrofluorimetric Protocol for Ceftriaxone in Commercial Formulation and Human Plasma After Condensation with Formaldehyde and Ethyl Acetoacetate

A new spectrofluorimetric method has been developed and validated for the quantification of ceftriaxone in bulk powder, pharmaceutical formulations and spiked human plasma. The developed method is reproducible, accurate, sensitive and cost effective. In this method, ceftriaxone was converted into a fluorescent compound by reacting with 0.8 M ethyl acetoacetate and 25% formaldehyde in a buffered medium (pH = 4.2) at 90 °C. The excitation and emission wavelengths of the fluorescent reaction product are 316 nm and 388 nm respectively. Optimization of the experimental conditions affecting the condensation reaction were carefully carried out and the optimum experimental conditions were incorporated in the procedure. The developed method has a broad linear range (0.2-20 μg mL(-1)) with a correlation coefficient of 0.9992. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 1.94 × 10(-2) μg mL(-1) and 6.47 × 10(-2) μg mL(-1) respectively. The common excipients and co-administered drugs were investigated for their interferences effect in the assay. The developed method was validated statistically through recovery studies and successfully applied to ceftriaxone determination in bulk powder, pharmaceutical formulations and spiked human plasma samples. The percent recoveries were found to be in the range of 99.04-100.26% for bulk powder, 98.88-99.92% for pharmaceutical formulations and 94.22-98.48% for spiked human plasma. The results were verified by comparing with reference literature HPLC method and were found in good agreement.

Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of β-galactosidase (β-gal) knockout ((-/-)) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (β-gal(+/-)) and GM1 gangliosidosis (β-gal(-/-)) mice. Total ganglioside content (μg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in βgal(-/-) mice than in βgal(+/-) mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (μg/mg protein) was similar in the PM and the MS fractions of the βgal(-/-) and βgal(+/-) mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the β-gal(-/-) mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.

Development of spectrofluorimetric methods for the determination of levosulpiride in pharmaceutical formulation

Simple, accurate, rapid, and sensitive spectrofluorimetric methods for the determination of levosulpiride in pharmaceutical formulation were developed utilizing its fluorescence reaction with Fe3+ (method A) and Al3+ (method B). The calibration curves were found to be linear in the concentration range 0.239–3.44 μg/mL and 0.310–2.730 μg/mL with limit of detection 0.005 μg/mL and 0.0032 μg/mL, respectively, for method A and method B. The reaction conditions were studied and optimized. In addition, the complexation of Mg2+ and Ca2+ was also studied. In all cases, an enhancement in fluorescence emission of levosulpiride upon formation of complex with metal ions was observed. A 2: 1 (drug: metal) stoichiometry for all the complexes was established. Benesi-Hildebrand method was applied for calculation of association constant at 25 and 35°C. The thermodynamic parameters obtained in this study revealed that the interaction process was spontaneous and mainly ΔS-driven.

Determination of memantine in rat plasma by HPLC‐fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 µm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.

Molecular cloning and mRNA expression of cathepsin C in white shrimp, Litopenaeus vannamei

The cDNA encoding cathepsin C from the haemocytes of Litopenaeus vannamei (designated LVcathepsinC) was cloned by rapid amplification of cDNA ends (RACE) techniques. The full length of LVcathepsinC cDNA was 2026 bp, with an open reading frame of 1356 bp, which encoded a polypepetide of 451 amino acid residues with a theoretical molecular weight of 50.76 kDa and an estimated isoelectric point of 6.01. LVcathepsinC showed high similarity to other organisms on performing blast analysis. Fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction was used to examine the expression of the LVcathepsinC gene in haemocytes of L. vannamei after the challenge of bacteria Vibrio alginolyticus. There was a clear time-dependent expression pattern of LVcathepsinC after the bacterial challenge, and the mRNA expression reached a maximum level at 4 h post challenge, and then returned to the control level after 8 h. The up-regulated mRNA expression of LVcathepsinC in L. vannamei after bacterial challenge indicates that the LVcathepsinC gene is inducible and may be involved in immune response.

Letters to the Editor

Abstract For some time it has been known that warming with concentrated sulphuric acid caused condensation of several natural æstrogens with the production of coloured solutions with varying fluorescence effects. Boscott1 has recently developed the phosphoric acid reaction of Finkelstein, Hestrin, and Koch2 for the detection and estimation of steroid and synthetic æstrogens. The need for a reaction to distinguish between tablets containing small amounts of the various steroid and synthetic hormones in general has led us to investigate the possibility of extending Boscott’s technique for this purpose, since it is known that the presence of small amounts of tablet disintegrants and lubricants interferes with certain colour reactions for these substances (cf. Cocking3). The basic technique described by Boscott was followed after preliminary extractions (when necessary) of the crushed tablets with ether and evaporation of the solvent. It is not certain in cases where extraction had to be used what accompanying tablet constituent was interfering with the fluorescence reaction; starch interfered with the dienæstrol reaction but in other cases merely caused a slight alteration of the fluorescence colour. The results given by a number of steroid hormones not previously examined are also recorded.

The expression of TLR2,4,7mRNA on peripheral blood mononuclear cells in patients with chronic cystic echinococcosisinfection

Objective To explore the expression of TLR2,4,7 mRNA on peripheralblood mono-nuclear cells (PBMCs) in patients with chronic cystic echinococcosis(CE)infection, and the level of serum IL-10. Methods The expression level of TLR2,4,7 mRNA onperipheral blood mononuclear were tested in 42 chronic CE cases and 28 normal controls(NC) by real-time fluorescent quantitative reverse transcription polymerase chain reaction(FQ-RT-PCR) method. GAPDH was selected as the internal control. The level of serum IL-10was determined in ELISA. The subjects were determined by t test. The correlations betweenTLR2, TLR4, TLR7 and IL-10 were determined by differences of expression of TLR2, TLR4,TLR7 on PBMCs and serum IL-10 in two groups of study linear correlation test. Results Theexpressions of TLR2, TLR4,TLR7 mRNA in chronic CE group were higher than those of in NCgroup. Compared with the NC group, the expressions of TLR2, TLR4 and TLR7 mRNA increasedmore than 7.3-, 3.6-, 3.6-fold, respectively. In chronic CE group, TLR2, TLR4 and TLR7mRNA expressions were 1.0729 ±0.4006, 5.0976 ±1.6682, 0. 6481 ±0. 2574, respectively.TLR2, TLR4 and TLR7 mRNA expressions were 0. 1468 ± 0.0435, 1.4067 ±0. 3279, 0. 1804 ±0.0568 in NC group, respectively. Compared with NC group, the differences of TLR2 and TLR4mRNA expression were significant (P = 0.0287, 0. 033), while the expression of TLR7 mRNAwas not difference (P =0.0862). Moreover, in chronic CE group, the level of serum IL-10was higher than that of in NC group. In chronic CE group and NC group, the level of serumIL-10 was (17.6770±1.6298) pg/ml, (9.4898 ±0.7049) pg/ml. Compared with NC group, therewas significant difference in chronic group (P<0.01). Significant positive correlationbetween TLR2 and TLR4 was found in chronic CE group, r = 0. 1135, P =0.036. Others werenot correlations. Conclusion In the development of chronic CE, TLR2 and TLR4 participatein this progression. As the receptors of antigen of cystic echinococcus, TLR2 and TLR4 canregulate the immune response through interacting with different antigens from cysticechinococcus. Meanwhile, under the participation of TLR2, TLR4 and increased serum IL-10,they will approach to Th2 immune reaction, which play an important role in chronic CE thatcan induce immune evasion.

The relationship of between HCV RNA and Anti-HCV

Objective To explore the HCV-RNA positive rate of the anti-HCV positive patients, and provide the diagnosis and treatment of HCV infection with evidence. Methods Thirty-three patients, who were Anti-HCV positive, were enrolled in this study. Use auantitafed fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) to test HCV RNA. Anti-HCV was defecfed by enzyme linked immunosorbent assay(ELISA).Results Among 33 patients,there were 22 cases with quantitation of HCV RNA higher than 80 copy/ml.The positive rate was 66.67%.67%.conclusion Simultaneusly testing the quantitation of HCV RNA and Anti-HCV is helpful for the early diagnosis and treatment of Hepatitis C infenction. Key words: Hepatitis C viru; Anti-HCV; PCR

Increased expression of microRNA-146a in peripheral blood mononuclear cells of patients with chronic immune thrombocytopenic purpura and its clinical significance

Objective To investigate the increased expression of microRNA-146a（miR-146a） in peripheral blood mononuclear cells （PBMC） of patients with chronic immune thrombocytopenic purpura （ITP） and its clinical significance. Methods Twenty-eight patients with chronic ITP and 28 healthy controls matched with age and gender were enrolled in this study. Fluorescent quantitative PCR reaction was used to detect the relative expression of miR-146a in their PBMC. The serum concentration of TNF-α, IL-2,IL-1 β and IFN-γ were measured by ELISA. CCK-8 method was used to detect the proliferation ability of PBMC , which transfected with miR-146a mimics or inhibitor and then stimulated with platelet . Results The relative expression of miR-146a in ITP patients was higher than that of healthy controls. The increased expression of miR-146a was negatively correlated with the serum TNF-α, IL-2 and IFN-γ. The PBMC transfected with miR-146a mimics had reduced expression of IL-2 and proliferation when stimulated with platelet.In contrast, the opposite effect was observed with the miR-146a inhibitors transfection. Conclusion MiR146a was involved in the pathogenesis of chronic ITP by controlling IL-2 production and PBMC proliferation.Thus, it may be a potential therapy target for chronic ITP. Key words: Chronic immune thrombocytopenic purpura MicroRNA-146a Autoimmune

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD + as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.

PARAFAC based methods for the analysis of Diltiazem drug excitation emission matrices of fluorescence obtained by a derivatization reaction

A fluorescent derivatization reaction for Diltiazem drug quantification based on the condensation reaction of citric or malonic acid with acetic anhydride, catalyzed by the tertiary amine group of Diltiazem, was developed. Excitation emission matrices (EEMs) of fluorescence of the pure solvent (ethanol), standard and sample solutions following a standard addition methodology were analysed by PARAFAC based methods (PARAFAC, PARAFAC2 and PARALIND) to obtain robust calibration methodologies. The quantification results of the sample were compared with the official US Pharmacopeia high performance liquid chromatography-ultraviolet method (USP HPLC-UV). Although the experimental sets of EEM show linearity deviations all the PARAFAC based methods allow correct robust estimation of Diltiazem concentration in pharmaceutical formulations. The closest results were: derivatization with citric acid and PARAFAC2 six components non-negativity constraint model with a detection limit of 0.088 ppm; and, derivatization with malonic acid and PARAFAC six components non-negativity constraint model with a detection limit of 0.066 ppm for the malonic acid was observed. The simultaneous utilization of the three PARAFAC methods gives further information about the intrinsic structure of the data sets under analysis, i.e., it works as an efficient diagnostic tool for the existence of non-linearity and colinearity.

Molecular strategies to read and write at the nanoscale with far-field optics

Diffraction prevents the focusing of ultraviolet and visible radiations within nanoscaled volumes and, as a result, the imaging and patterning of nanostructures with conventional far-field illumination. Specifically, the irradiation of a fluorescent or photosensitive material with focused light results in the simultaneous excitation of multiple chromophores distributed over a large area, relative to the dimensions of single molecules. It follows that the spatial control of fluorescence and photochemical reactions with molecular precision is impossible with conventional illumination configurations. However, the photochemical and photophysical properties of organic chromophores can be engineered to overcome diffraction in combination with patterned or reiterative illumination. These ingenious strategies offer the opportunity to confine excited chromophores within nanoscaled volumes and, therefore, restrict fluorescence or photochemical reactions within subdiffraction areas. Indeed, information can be "read" in the form of fluorescence and "written" in the form of photochemical products with resolution down to the nanometre level on the basis of these innovative approaches. In fact, these promising far-field optical methods permit the convenient imaging of biological samples and fabrication of miniaturized objects with unprecedented resolution and can have long-term and profound implications in biomedical research and information technology.

Study on the Expression of Organic Anion Transporting Polypeptide (oatp2a1) in Rat with Spleen Deficiency Syndrome and the Exploration of Clinical Significance

Objective: to explore the mechanism of transportation and transformation of dampness by the way of the expression of organic anion transporting polypeptide (oatp) superfamily member 2a1 (oatp2a1) mRNA in rat with spleen deficiency syndrome and the significance in transportation and transformation of dampness. Methods: 32 wistar male rats were divided randomly into four groups: normal group (n = 6), normal + AA group (n = 6), spleen deficiency group (n = 10), Spleen deficiency + AA group (n = 10). After reserpine-induced spleen deficiency model was made, intragastric administration of aristolochic acid (AA) was adopted for three days, the expression of oatp2a1 mRNA were detected in the tissues of lung, liver, kidney, stomach, small intestine and large intestine in four groups by using Fluorescent Quantitative-Polymerase Chain Reaction (FQ-PCR). Results: the expression of oatp2a1 mRNA in above six tissues could be detected. The ex-pression of oatp2a1 mRNA in liver tissue of rat with spleen deficiency syndrome was up-regulated compared to normal group (P = 0.035, P < 0.05), the expression of oatp2a1 mRNA in small intestinal tissue of rat with spleen deficiency syndrome was down-regulated compared to normal group (P = 0.004, P < 0.01), the expression of oatp2a1 in intestinal tissue in normal + AA group is down-regulated compared to normal group (P = 0.032, P < 0.05). Conclusions: oatp2a1 might be one of the material basis involved in transportation and transformation of dampness. The changes of expression of oatp2a1 mRNA in small intestine, liver tissue suggests that small intestine, liver might play an important role in the transportation and transformation of dampness in the state of spleen deficiency. We further concluded that the function of spleen’s governing transportation and transformation of dampness was not only including the function of the gastrointestinal, but also part of the liver function in some degree, which needs to be further studied.

Effects of Chinese herbal medicine Yiqi Huoxue Formula on TGF-β/smad signal transduction pathway and connective tissue growth factor in rats with renal interstitial fibrosis

To observe the effects of Yiqi Huoxue Formula (YQHXF), a compound Chinese herbal medicine, on transforming growth factor-β (TGF-β)/smad signal transduction pathway and connective tissue growth factor (CTGF) in rats with renal interstitial fibrosis Unilateral ureteral obstruction (UUO) rat model was established and the rats were randomly divided into 5 groups: untreated group, high-, medium-, and low-dose YQHXF groups and fosinopril sodium group. Another group with sham operation was set as control. All rats were administered with corresponding drugs for 3 weeks. After the last administration, each rat was sacrificed and weighed and the serum was separated for creatinine (Cr) and blood urea nitrogen (BUN) detection. Kidneys of the rats were taken out, and mRNA and protein expressions of TGF-β, smad2, smad7 and CTGF were measured with real-time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting respectively; fibrosis of the kidney tissue was observed with hematoxylin-eosin (HE) staining and Masson trichrome staining. Compared with sham-operation group, Cr and BUN in serum of UUO groups were increased, while high-dose YQHXF treatment decreased the UUO-induced increase of Cr and BUN levels. HE staining and Masson staining results showed that the renal tubular epithelial cells in untreated group got atrophied; lumens of renal tubules expanded; fibroplastic proliferation and inflammatory cell infiltration were observed in renal interstitium; the number of glomerulus decreased and collagen increased significantly compared with sham-operation group. In the high- and medium-dose YQHXF groups and fosinopril sodium group, the histopathological changes of inflammatory cell infiltration, fibroplastic proliferation, expansion of lumens of renal tubules was improved as compared with the untreated group. The mRNA and protein expressions of TGF-β, smad2 and CTGF in untreated group were higher than those in sham-operation group (P<0.05), and the mRNA and protein expressions of smad7 in untreated group were lower than those in the sham-operation group (P<0.05). Compared with untreated group, high- and medium-dose of YQHXF significantly down-regulated the mRNA and protein expressions of TGF-β, smad2 and CTGF (P<0.01, P<0.05), and up-regulated the mRNA and protein expressions of smad7 (P<0.01, P<0.05). The mRNA expression of CTGF in UUO rats may be regulated by TGF-β/smad signaling transduction pathway. YQHXF might inhibit the expression of CTGF through down-regulation of TGF-β and smad2 and up-regulation of smad7, thus inhibiting the progression of renal interstitial fibrosis.

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.

Spectrofluorimetric Method for Determination and Validation of Cefixime in Pharmaceutical Preparations Through Derivatization with 2-Cyanoacetamide

A simple, sensitive and accurate method has been developed for spectrofluorimetric determination of cefixime in pure form and pharmaceutical preparations. The method is based on the reaction of cefixime with 2-cyanoacetamide in the presence of 21% ammonia at 100 °C. The fluorescent reaction product showed maximum fluorescence intensity at λ 378 nm after excitation at λ 330 nm. The factors affecting the derivatization reaction were carefully studied and optimized. The fluorescence intensity versus concentration plot was rectilinear over the range of 0.02 to 4 μg mL(-1) with correlation coefficient of 0.99036. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 2.95 ng mL(-1) and 9.84 ng mL(-1), respectively. The proposed method was validated statistically and through recovery studies. The method was successfully applied for the determination of cefixime in pure and dosage form with percent recoveries from 98.117% to 100.38%. The results obtained from the proposed method have been compared with the official HPLC method and good agreement was found between them.

In vitroeffect of nickel on bovine spermatozoa motility and annexin V‐labeled membrane changes

In this study the effect of in vitro culture of bovine spermatozoa with nickel (NiCl(2)) on spermatozoa motility and membrane changes was analyzed. The spermatozoa motility significantly decreased after 120 min of culture at the concentration of 1000 μM Ni ml(-1) (P < 0.05) and after 240 min of culture at the concentration of 500 and 1000 μM Ni ml(-1) (P < 0.001) as compared with control. The progressive motility was the highest in the control group and in the groups with the lowest nickel concentrations (7.8 and 125 μM Ni ml(-1)). The progressive spermatozoa motility was significantly altered even after 30 min of culture in the group with the highest nickel concentration (1000 μM Ni ml(-1)). A significant decrease in progressive motility from the concentration of 250 μM Ni ml(-1) was detected after 240 min of culture. Concentrations from 125 μM Ni ml(-1) in various time periods of culture stimulated spermatozoa motility after 30 min (P < 0.001), but later an inhibitory effect was noted. After 240 min of in vitro spermatozoa culture with 125 μM Ni ml(-1) a typical Annexin V fluorescence reaction was detected. Fluorescence was detected in mitochondrial segment of bovine spermatozoa. In spermatozoa exposed to higher nickel concentrations the Annexin V-positive reaction was detected also on the spermatozoa head membrane. In the group with the highest concentration and the longest time of exposure (1000 μM Ni ml(-1); 240 min) the apoptotic Annexin-positive regions were detected not only in the mitochondrial part, but also in the spermatozoa head (acrosomal and postacrosomal part), showing significant alteration of spermatozoa membrane integrity.