Discontinuous Density Gradient Research Articles

Increased activation of nuclear factor-kappa B (NF-κB) in isolated peritoneal macrophages of patients with endometriosis

Endometriosis is characterized by pelvic inflammation that shows an increased number of activated peritoneal macrophages and their secreted products such as cytokines, growth factors, and angiogenic factors. Our results show that activation of nuclear factor-kappa B (NF-kappaB), a pro-inflammatory transcription factor, is statistically significantly increased in peritoneal macrophages from patients with endometriosis when compared with controls.

Biochemical separation of Na+,K+-ATPase from a "purified" light density, "canalicular"-enriched plasma membrane fraction from rat liver.

Cytochemical studies suggest that Na+,K+-ATPase is localized to sinusoidal and lateral portions of the hepatocyte plasma membrane whereas Mg++-ATPase and alkaline phosphatase are luminal or canalicular membrane markers. To validate further these cytochemical findings, we have isolated from the nuclear pellet of rat liver homogenates a liver plasma membrane (LPM) fraction enriched in all three enzyme markers, as previously described (Biochimica et Biophysica Acta 1975; 401:59-52). Following tight Dounce homogenization, the vesiculated membrane preparation was further separated on a multiple-step discontinuous sucrose density gradient (d 1.12 to 1.22). Na+,K+-ATPase activity "dissociated" from Mg++-ATPase activity, sedimenting at densities of 1.14 and greater. Further studies were carried out in two-step discontinuous sucrose gradients (1.13 and d greater than 1.13), and a light density fraction (d 1.13) was further characterized in calcium-free media (since addition of calcium increased contamination with intracellular membranes). Electron microscopy demonstrated a homogeneous vesicular membrane population in contrast to the heavy density fraction (d greater than 1.13) which contained membrane sheets and junction complexes as well as vesicles. The light density fraction was highly enriched n Mg++-ATPase (42.1 x homogenate specific activity) and alkaline phosphatase (64.6 x homogenate), 3 to 4 times their activities in the original LPM. In contrast, Na+,K+-ATPase activity in the light density fraction, diminished 16-fold from values in the original unfractionated LPM. All but 15% of total Na+,K+-ATPase activity in the original LPM could be accounted for in unwashed preparations. Neither cholesterol/phospholipid ratios nor an analysis of peptides on sodium dodecyl sulfate gel electrophoresis demonstrated differences in the composition of the light vs. heavy density subfractions, although there were relative increases in several peptide bands in the light density subfraction. These studies provide further supporting biochemical evidence for the concept that Na+,K+-ATPase resides on different membrane domains than does Mg++-ATPase and alkaline phosphatase and further characterizes a vesiculated membrane preparation highly enriched in putative "canalicular" enzyme markers.

Human glycolipid transfer protein—Intracellular localization and effects on the sphingolipid synthesis

Glycolipid transfer proteins (GLTPs) are small proteins that specifically transfer glycolipids from one bilayer membrane to another in vitro. However, the precise biological function is still unknown. In this study the intracellular distribution of GLTP was determined. We have used several independent methods, including differential and discontinuous density gradient centrifugation, plasma membrane permeabilization and confocal microscopy imaging, and we demonstrate that GLTP has a cytosolic location. The GLTP is not located in the Golgi apparatus, endoplasmic reticulum, nucleus, lysosomes, mitochondria or peroxisomes in HeLa cells. We have also used a fluorescence resonance energy transfer assay to detect transfer of fluorescently labeled BODIPY-glucosylceramide in the cytosolic fraction from both wild-type and GLTP-overexpressing HeLa cells. Furthermore, we have studied de novo sphingolipid changes in cells overexpressing GLTP using sphinganine metabolic labeling. The results show a significant increase in the synthesis of glucosylceramide (GlcCer) and a decrease in the sphingomyelin (SM) synthesis. However, no changes were detected in the de novo sphingolipid synthesis in GLTP-knockdown cells compared to control cells. We propose that GLTP is not likely involved in the de novo synthesis of glycosphingolipids, but could rather have a role as a glycolipid sensor for the cellular levels of glucosylceramide.

Kv11.1 (ERG1) K+ Channels Localize in Cholesterol and Sphingolipid Enriched Membranes and Are Modulated by Membrane Cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) α -subunit protein, which underlies the rapidly activating, delayed rectifier K+ current (IKr) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-Mβ -cyclodextrin (Mβ CD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (IKv11.1). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of IKv11.1 activation and accelerated the inactivation kinetics of IKv11.1. Incubation of neonatal mouse myocytes in Mβ CD also accelerated the deactivation kinetics of IKr. We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate IKv11.1 and IKr.

Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

Bacillus thuringiensis BT-1, BT-2가 생산하는 δ-endotoxin의 특성 규명

Bacillus thuringiensis is a well-known species of entomophathogenic bacteria that is widely used as a biopesticide against many insect pests. It produces parasporal crystals (δ-endotoxin) and endospores during sporulation. In this report, the δ-endotoxin produced by Bacillus thuringiensis BT-1 and BT-2 were characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), SDS-PAGE, and solubilization activity by alkaline solution. BT-1, BT-2 were cultured in the GBY medium, and the δ-endotoxin of them was purified with discontinuous sucrose density gradient centrifugation. Their δ-endotoxin was observed by SEM and TEM. Morphologically, the δ-endotoxin of BT-1 was a square and flat type, whose size was 1.73 ㎛ × 0.7 ㎛, and the δ-endotoxin of the BT-2 was spherical form whose size was 1.1 ㎛ × 0.9 ㎛ determined by SEM and TEM. The δ-endotoxin of the BT-1 was composed of 28 kDa and 21 kDa, however, it of the BT-2 was composed of 50 kDa, 35 kDa, and 22 kDa bands determined by SDS-PAGE. The purified crystals of BT-1 and BT-2 were dissolved gradually in alkaline solution as time goes by, and it was perfectly dissolved after 3 hours. It is supposed that the δ-endotoxin of crystal was converted to a state of activation in the course of time in the intestines of insect.

Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

The vasoconstrictor angiotensin II (Ang II) acts at G(q/11)-coupled receptors to suppress ATP-sensitive potassium (K(ATP)) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K(ATP) channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels. We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K(ATP) currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCepsilon translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K(ATP) channels by Ang II arises exclusively from the activation and translocation of PKCepsilon. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCepsilon to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K(ATP) channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCepsilon to these membrane compartments. Ang II induces the recruitment of the novel PKC isoform, PKCepsilon, to arterial smooth muscle caveolae. This translocation allows PKCepsilon access to K(ATP) channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

RNA dynamics of fertile and infertile spermatozoa

The presence of a complex population of mRNAs in human mature spermatozoa is well documented; among them, transcripts of aromatase and ERs (oestrogen receptors) have been described but their significance is not clear. Therefore, to clarify the role of this complex population of mRNAs in human ejaculated sperm, we have isolated on discontinuous density gradients two main fractions from the same sample: high- and low-motile spermatozoa. The levels of different transcripts coding for molecules involved in nuclear condensation [Prm-1 (protamine 1) and Prm-2], capacitation [eNOS (endothelial nitric oxide synthase), nNOS (neuronal nitric oxide synthase), c-myc], motility and sperm survival (aromatase) have been assessed using semi-quantitative RT (reverse transcriptase)-PCR. The viability of sperm as well as the percentage of apoptosis were identical in high- and low-motile fractions. No significant change in the c-myc/Prm-2 ratio between the two populations of spermatozoa was observed. Conversely the amount of Prm-1 mRNA was significantly higher in low-motile than in high-motile fraction; in most of the high-motile sperm samples analysed, eNOS and nNOS transcripts were undetectable, whereas they were observed in low-motile sperm. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. As to the aromatase expression, a significant decrease in the amount of transcripts in immotile sperm fraction was recorded in all samples studied. To conclude, analysing mRNA profiles in humans could be helpful either as a diagnostic tool to evaluate male fertility, since they reflect spermatogenesis gene expression, and/or a prognosis value for fertilization, since these RNAs are delivered to oocytes.

Semen processing by density gradient centrifugation does not improve sperm apoptotic deoxyribonucleic acid fragmentation rates

We wished to verify whether semen processing by discontinuous double-layered density gradient centrifugation could improve sperm apoptotic DNA fragmentation rates using a commercially available deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay in 35 consecutive men presenting for assisted reproductive treatments. Although sperm motility did improve as expected, no effects were observed in sperm apoptotic DNA fragmentation rates, and this should be considered in the routine assisted reproduction setting.

Low‐density lipoprotein receptor‐related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway

The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid beta protein (Abeta), a small peptide derived from beta- and gamma-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that beta- and gamma-secretase activities of BACE1 and presenilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno-precipitation assays, we show that the cytoplasmic domain of low-density lipoprotein receptor-related protein (LRP) interacts with APP and increases Abeta secretion and APP beta-CTF (C-terminal fragment) generation by promoting BACE1-APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain-mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where beta- and gamma-secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Abeta generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Abeta generation by targeting LRP-mediated delivery of APP to raft microdomains.

Simultaneous quantification of pathogenic Campylobacter and Salmonella in chicken rinse fluid by a flotation and real-time multiplex PCR procedure

A procedure for simultaneous quantification of Campylobacter and Salmonella spp. in poultry skin rinse fluids by a flotation and real-time multiplex PCR method is described. Flotation of the target organisms in a discontinuous density gradient separated them from background microflora, particles from poultry skin, dead target cells and PCR inhibitors. Variation of the buoyant density between 1.052 to 1.106 g/ml was measured at different times for various Salmonella strains grown over a period of 4 weeks. This, and the results from earlier studies on the buoyant densities of Campylobacter spp., which were between 1.065 and 1.109 g/ml, led to design of an optimal discontinuous flotation method with three density layers, of 1.048, 1.109 and approximately 1.200 g/ml. This method preceded a real-time multiplex PCR assay using hybridization probes. The specificity of the PCR assay was confirmed on 73 target and non-target strains, and target organisms were detected at the level of one genome per PCR. Results obtained with the combined flotation and real-time multiplex PCR method showed that quantification in rinse fluids was possible down to 3.0 ± 0.3 × 10 3 CFU/ml in the presence of other microorganisms at numbers up to 10 9 CFU/ml.

Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord

Neuron–glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep™. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.

Anionic Lipid Interaction Induces Prion Protein Conformational Change

The conversion of the prion protein (PrP) to the pathogenic PrPScconformation plays a central role in prion disease. However, the precise mechanism underlying this process remains unclear. Here, we report the conformational conversion of PrP upon interaction with anionic lipids. After the discontinuous iodixanol density gradient centrifugation, we found strong binding between PrP and negatively charged phospholipids, involving both electrostatic and hydrophobic interactions. Under physiologically relevant conditions, interactions with lipid were sufficient to convert full-length, α helices rich recombinant mouse PrP to a conformation similar to PrPSc, with increased β sheet content and a PrPSc-like proteinase K (PK)-resistant pattern. Conversion is greatly influenced by lipid headgroup structures and lipid vesicle compositions. When lipid vesicles are disrupted by detergent, aggregation is necessary to maintain the PK resistant conformation. Our results imply that the strong lipid-PrP interaction is sufficient to overcome the energy barrier between the two conformational states and support the notion that lipid membrane may play a role in PrP conformational change.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets inin vitroculture

To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Quantitative Assessment of Transition Proteins 1, 2 Spermatid-Specific Linker Histone H1-Like Protein Transcripts in Spermatozoa from Normozoospermic and Asthenozoospermic Men

Spermatid-specific linker histone H1-like protein (HILS1), transition proteins 1 and 2 (TNP1 and TNP2), and protamines 1 and 2 (PRM1 and PRM2) contribute to considerable dense packing of spermatid chromatin during spermiogenesis. We evaluated the HILS1, TNP1, and TNP2 transcript levels in spermatozoa isolated from normozoospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n = 70) and asthenozoospermic (n = 100) donors were purified by centrifugation through a discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczynski and Sacchi method, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of HILS1, TNP1, and TNP2 transcripts was performed by real-time quantitative (RQ-PCR) SYBR green I analysis. We found significantly lower levels of HILS1, TNP1, and TNP2 transcripts in spermatozoa from asthenozoospermic men compared to normozoospermic men. Our observations suggest that a reduction in HILS1, TNP1, and TNP2 transcripts may be associated with asthenozoospermia.

Adenylyl cyclase 5/6 colocalizes with TRPC4 and cell adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell-cell junctions between adjacent pulmonary capillary endothelial cells contribute to the integrity of this barrier and are dynamically regulated by transitions in cAMP. Within PMVECs calcium sensitive adenylyl cyclase activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of adenylyl cyclase and other signaling molecules involved in barrier regulation within the PMVEC membrane has not been described. Here we utilize a detergent free discontinuous sucrose density gradient centrifugation technique to separate caveolae/lipid raft fractions from the bulk plasma membrane. AC5/6 immunoreactivity was detected in the caveolin rich light-density fractions while beta adaptin immunoreactivity was detected in the high-density fraction of non-caveolae regions within the plasma membrane. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in the caveolin rich fraction. In addition, cell adhesion molecules N-cadherin and ALCAM and the scaffolding molecule Dlg localize to caveolae. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and HL60024

Staphylococcus aureus Subvert Autophagy for Induction of Caspase-independent Host Cell Death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.

An Improved Flow Cytometric Approach for Isolation of Fetal Cells from Maternal Blood for Non Invasive Prenatal Diagnosis of Hemoglobinopathies

Prenatal diagnosis is an option for couples at risk of having a child affected with hemoglobinopathies. Chorionic villus sampling (CVS) and cordocentesis are accurate but a finite risk of fetal loss exists. A non invasive, risk free strategy that has emerged is isolation of fetal erythroblasts from maternal blood. Enrichment of nucleated red blood cells (nRBCs) from 7.0 mL maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies: CD45 per CP, glycophorin A-phycoerythrin (PE) and Hb F-fluorescein isothiocyanate (FITC) in 43 cases between 7 and 21 weeks' gestation. The percentage of nRBCs ranged from 0.0001–2.03%. The presence of dual fluorescence (glycophorin A-PE and Hb F-FITC) was confirmed by confocal microscopy. A sufficient number of nRBCs could be isolated in the first and second trimester of pregnancy to provide a simple flow cytometric approach as a potential for non invasive diagnosis of β-globin defects.

Detection of fetal nucleated red blood cells in the maternal circulation by Kleihauer test

Maternal blood was obtained from 18 pregnant women at 7 to 25 weeks of gestation. After Percoll discontinuous density gradient centifugation, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. Positive fetal cells appeared with an intense red cytoplasmic staining while maternal cells with adult haemoglobin were colourless. Individual positive NRBC was collected by micromanipulator and whole genome amplification was then performed to determine sex and STR status. This allowed the simultaneous verification of the fetal origin of NRBC and prenatal diagnosis of genetic diseases. The non-invasive prenatal genetic diagnosis of 9 fetuses at high risk of Duchenne muscular dystrophy (DMD) was completed successfully. The Kleihauer test is a rapid, simple and direct chemical staining method to select fetal cells and can be applied in prenatal diagnosis.

Spermicidal activity of extract from Cestrum parqui

Objective The leaf extract of Cestrum parqui was examined for its effects on sperm motility in vitro. Methods Washed sperm were prepared by discontinuous buoyant density gradient centrifugation and incubated with varying concentrations of extract from C. parqui (40–250 μg/mL) at 37°C and 5% CO 2. Sperm motility and viability were assessed at different intervals ranging from 5 to 240 min. Morphological changes in human ejaculated spermatozoa after exposure to the extract were evaluated under transmission electron microscope. Results A dose- and time-dependent effect of this extract on sperm motility and viability was observed. The maximal spermicidal effect was observed with a 250-μg/mL dose of the extract. Transmission electron microscope revealed a significant damage to sperm membrane in both head and acrosomal membranes, notable swelling and disruption. Conclusion The present study indicates that this natural extract has potential spermicidal effect in vitro.