Combination Of Density Gradient Centrifugation Research Articles

P-031 Viral crossroads: revisiting sperm processing in the context of Human Immunodeficiency Virus (HIV) infection

Abstract Study question Is extensive sperm washing, followed by viral load testing, necessary to ensure safety of assisted reproductive techniques in HIV-positive men? Summary answer In context of viral suppression, there is no added value of extensive sperm washing followed by viral load testing for HIV-positive men seeking fertility treatment. What is known already Sperm washing was initially introduced as a safe reproductive strategy for serodiscordant couples in which the male partner is seropositive. This technique uses a combination of density gradient centrifugation (DGC) and swim-up (SU) to separate the HIV-free spermatozoa from the seminal plasma containing most of the viral particles. Nowadays, due to the increased availability and efficacy of antiretroviral drugs, more patients are adequately suppressed and have no risk of transmitting HIV, which is reinforced by the ‘Undetectable Equals Untransmittable’ principle. This questions the need for extensive sperm washing followed by quantitative polymerase chain reaction (qPCR) testing in the IVF laboratory. Study design, size, duration This single center retrospective study investigates the effectiveness of our existing sperm washing procedures by evaluating the HIV viral load (VL) both in the raw and processed semen fractions, while considering the patient’s viral suppression status. All sperm wash procedures performed on fresh ejaculates between 2012 and 2023 were included. Additionally, clinical outcomes of the subsequent intracytoplasmic sperm injection (ICSI)-cycles were collected. Participants/materials, setting, methods Type 1 (wash-DGC-SU), type 2 (wash-DGC) or type 3 (double wash) sperm processing was executed based on the concentration and motility of the raw sample. The processed fraction was cryopreserved, and the VL was determined in both the raw and processed semen fraction using qPCR. A semen VL below 20 copies/mL was considered negative. Only processed fractions with a negative VL were suitable for further therapeutic use. Threshold for viral suppression was 50 copies/mL. Main results and the role of chance A number of 82 sperm processing procedures were performed for 68 seropositive men, with a mean age at donation of 41.9 ± 7.0 years. All patients were confirmed to be HIV-1 positive, with the exception of one individual presenting a co-infection with both HIV-1 and HIV-2. Incidences for type 1, type 2 and type 3 processing were 23.2%, 62.2% and 14.6%, with mean initial concentration of 91.3(±63.6), 42.1(±47.9) and 0.12(±0.15) million/mL, and mean progressive motility of 54(±12.7)%, 33(±16.8)% and 4(±3.6)% in the respective groups. In 91.5% (n = 75/82) of cases, the patient was suppressed at the time of sperm processing. In case of viral suppression, only one raw sample (n = 1/75, 1.3%) tested positive for HIV (94 copies/mL), likely due to the phenomenon of “viral shedding”. After processing, none of the fractions contained a residual VL. Post-thaw progressive motile sperm survival rate was on average 0.40 ± 0.26. Overall, 61 couples underwent a total of 120 ICSI-cycles, resulting in cumulative clinical pregnancy and cumulative live birth rates per couple of 57.4% (n = 35/61) and 42.6% (n = 26/61), respectively. Limitations, reasons for caution Given that HIV-positive individuals represent a minority within our patient population, the limited prevalence of sperm wash procedures performed over the past decade has led to a relatively small sample size for this study. Wider implications of the findings These findings have the potential to shape future guidelines concerning the use of sperm samples from HIV-positive men. This may optimize the efficiency of sperm processing protocols, while maintaining both the safety and efficacy of assisted reproductive techniques for this specific patient population. Trial registration number Not applicable

Density Gradient Centrifugation Alone or the Combination of DGC with Annexin V Magnetic-Activated Cell Sorting Prior to Cryopreservation Enhances the Postthaw Quality of Sperm from Infertile Male Patients with Poor Sperm Quality

Objective. To examine whether density gradient centrifugation (DGC) alone or its combination with annexin V magnetic-activated cell sorting (DGC-MACS) can be used to process semen samples from infertile male patients with poor sperm quality prior to subjecting these to freeze/thaw process in order to optimize the outcomes of sperm freezing. Methods. This study enrolled sixteen patients with sperm concentration ≥ 20 × 10 6 / mL , sperm motility < 30 %, and/or <4% normal sperm morphology. Sperms were processed by DGC or DGC-MACS prior to the freeze/thaw process. Sperm motility, hyperosmotic swelling test (HOS), TUNEL test, and morphological analysis were performed before and after the freeze/thaw process. Results. The freeze/thaw process had a detrimental effect on sperm motility, viability, morphology, and DNA integrity in all three groups (RAW, DGC, and DGC + MACS groups). The DGC and DGC + MACS groups showed increased sperm motility, viability, and normal morphology following freeze/thaw than untreated frozen controls. The motility and viability were not significantly different between DGC-MACS-CPT (cryopreservation-thawing) and DGC-CPT groups. Moreover, almost no grade A or grade B sperm was observed in the DGC-MACS-CPT groups. The sperm selected by DGC or DGC + MACS showed decreased levels of sperm DNA fragmentation than RAW samples following freeze/thaw. Moreover, the sperm DNA fragmentation following freeze/thaw in the DGC-MACS-CPT group was significantly lower than that in the DGC-CPT group. Conclusions. Sperm preparation by DGC before cryopreservation improved the quality of sperm postthaw in infertile males with poor sperm quality. If the sperm quality following freeze/thaw is foreseen to be insufficient for artificial insemination with husband’s sperm or in vitro fertilization, or if there is high DNA fragmentation in RAW sperm, DGC + MACS should be used prior to cryopreservation to reduce sperm DNA fragmentation and improve the quality of sperm available for intracytoplasmic sperm injection.

Comparison between density gradient centrifugation method, an extended version of the horizontal swim up method and the combination of both for sperm selection.

To compare the degree of efficiency between density gradient centrifugation (DGC) method and an extended horizontal swim-up (SU) method. A total of 97 couples undergoing in vitro fertilization were enrolled in the study. Semen samples were divided into three aliquots and treated using DGC, extended horizontal SU, and combined methods. DNA fragmentation and chromatin decondensation were detected in native semen samples and their three corresponding aliquots. The corresponding mature oocytes of each semen sample were divided into two sibling cultures. The first sibling culture was microinjected with semen pellets from DGC, and the second sibling culture was microinjected with semen pellets from the combination of both methods. Fertilization rate and embryonic development were assessed at day 3. DNA fragmentation and chromatin decondensation was significantly low in DGC and extended horizontal SU samples; however, the rates of DNA fragmentation and chromatin decondensation were significantly lower in extended horizontal SU samples than in DGC samples. The lowest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with both methods. The highest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with DGC. No significant difference was found in the fertilization rate or day 3 embryos between sibling cultures. The combination of DGC and the extended horizontal SU techniques is best for giving the lowest rates of sperm DNA fragmentation and chromatin decondensation.

Changes in human sperm motility and DNA fragmentation index after incubation at different temperatures following density gradient centrifugation and swim-up procedures

Objective: The purpose of this study was to evaluate the sperm motility and DNA integrity at different temperatures to analyze whether the sperms are suitable on the second day for insemination of in vitro matured oocytes by intra-cytoplasmic sperm injection (ICSI) following density gradient centrifugation (DGC) and swim-up (SU) procedures. Methods: Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses. Following sperm selection by DGC and SU procedures, the liquified semen samples were divided into three groups and incubated at 4, 25, and 37°C, respectively. Following incubation for 24, 48, and 72 hours, the sperm motility and sperm DNA fragmentation index (DFI) were analyzed. Results: Following the combination of DGC and SU procedures, the sperm motility (91.8% ± 8.6% vs. 50.8% ± 13.1%) and DFI (5.1% ± 7.9% vs. 13.0% ± 11.6%) were significantly improved (P <0.01) compared to those without any treatment. The sperm motility of the 3 groups significantly declined (P <0.05) post-incubation compared to that of the groups prior incubation. However, sperm motility significantly increased (76.9% ± 10.4%) (P <0.05) at 25°C compared to that of the other 2 groups (53.5% ± 11.0% and 47.6% ± 10.2%). Sperm DFI significantly increased (P <0.05) at 37°C following incubation for 24 and 72 hours in comparison to that of the other 2 groups. However, the sperm DFI did not significantly increase when the sperm samples were incubated at 4 (5.7% ± 5.9%) and 25°C (6.8% ± 5.6%) for 24 hours compared to that before incubation (5.1% ± 7.9%). Conclusions: These results indicate that the sperm quality, in terms of motility and DFI, can be efficiently improved by DGC in combination with SU. Following which, the sperm samples can be incubated at 25°C and be used on the second day for insemination of in vitro matured oocytes by ICSI.

Elimination of bacteria from semen using a combination of density gradient centrifugation and swim-up

Background: To evaluate the efficiency of bacterial clearance of different sperm preparation procedure. Methods: A total of 46 semen samples were collected in this study, each semen sample was divided into four equal parts (0.5 mL each). The first part was prepared by density-gradient centrifugation (D-group), the second part was prepared by swim-up (S-group), the third part was treated with density-gradient centrifugation combined with swim-up (D+S-group), and the fourth group was the original raw semen group (O-group) regarded as the control. After each semen preparation procedure, the O-group and suspensions were inoculated and incubated for bacterial identification and colony counting. Results: Initially only 8.7% (4/46) of samples were bacteria free. After processing, the bacterial clearance rates were 23.8% for D-group, 57.4% for S-group and 97.8% for D+S-group (p < 0.001). Multiple bacterial strains were observed in 37 samples with 117 different bacterial strains in all identified in the original semen. 44.4% of the staphylococci and 89.7% of streptococci were not eliminated in D-group. In S-group, the corresponding rates were 24.4% and 35.9%. In D+S-group there were no remaining strains of staphylococci and only 2.6% of streptococci remained. After the combined procedure, the number of bacterial colonies fell dramatically after processing. Conclusion: The combined D+S protocol appears to be substantially more efficient than either method alone in eliminating bacteria from semen samples.

Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating.

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30–35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Zeta method: A noninvasive method based on membrane charge for selecting spermatozoa expressing high level of phospholipaseCζ.

Intracytoplasmic sperm injection (ICSI) is a technique developed to help attain successful fertilisation for couples with severe male factor. However, a small percentage of couples confront low or failed fertilisation, mainly due to failed oocyte activation. Several studies have introduced phospholipase Cζ (PLCζ) as the main sperm factor inducing oocyte activation and thereby has the potential to act as a biomarker for the prediction of ICSI fertilisation outcome. On the other hand, researchers have focused on novel sperm selection procedures based on cellular characteristics of spermatozoa such as surface electrical charge (Zeta potential) to isolate normal sperm subpopulation with intact chromatin. Therefore, we aimed to compare PLCζ between Zeta method and routine sperm preparation procedure: density gradient centrifugation (DGC). Our results showed that number of PLCζ-positive spermatozoa was significantly low in the Zeta method, but the intensity of PLCζ protein in such spermatozoa was significantly higher than DGC procedure. Therefore, the combination of DGC with Zeta procedure may allow selecting the population of spermatozoa with a high percentage of PLCζ which may also contain a high amount of PLCζ and with intact chromatin. This sperm selection procedure can open a new approach for infertile men with previously failed fertilisation.

IMPACT OF PREPARATION USING CONVENTIONAL AND MODIFIED DENSITY GRADIENT CENTRIFUGATION METHODS ON SPERM CONCENTRATION, MOTILITY AND NUMBER OF NORMAL MOTILE SPERM RECOVERY (NMSR)

Some preparation techniques, either conventional or advanced, have been provided. Advanced technique may overcome the limitations of conventional techniques. Recently, not all fertility clinics in Indonesia are able to provide advanced preparation techniques. Some techniques require expensive equipments and can only be used for intracytoplasmic sperm injection (ICSI). Some fertility clinics use a modified procedure, namely the combination of density gradient centrifugation with a swim-up method for the preparation of the sperm to be used in ART. This study aimed to determine whether the modified density gradient centrifugation, which is density gradient centrifugation followed by a swim-up, is able to yield better results than conventional density gradient centrifugation. This study was a laboratory experimental pre and pos-test control group design. Population was all adult men aged 21-40 years old and the sampling unit was the man donor’s semen which fulfilled inclusion criterias, collected during the periode of the study. Sample size was eight. Sperm analysis were done before and after preparation in conventional and modified group. Descriptive comparation analysis have been used. This study obtained NMSR 7.9+5.5 million/ejaculate and recovery rate (RR) 27.66+11.8 %. RR was lower compared to RR obtained conventional DGC method. RR might be lower because in modified DGC samples undergo two steps selection while conventional DGC only one step selection. But conventional DGC samples should be centrifuged twice, therefore sperms might experience more trauma. Lower RR sperm does not exclude the possibility to be used for ART, because still within the required number for all TRBs. In conclusion, modified DGC preparation method obtained lower NMSR and RR, nevertheless harvested sperms can still be used in all kind of ART.

Magnetic cell sorting of semen containing spermatozoa with high DNA fragmentation in ICSI cycles decreases miscarriage rate

This retrospective cohort study investigated whether reproductive outcome could be improved in couples presenting with a high level of sperm DNA fragmentation (SDF) by treating the ejaculate with the magnetic cell sorting (MACS) sperm selection procedure in combination with prior density gradient centrifugation (DGC). Only men presenting with ≥30% sperm DNA in the ejaculate were included because these patients can be potentially treated with MACS to reduce the proportion of sperm presenting DNA damage. In total, 305 couples were included in this study, and from these, 216 women underwent autologous ICSI (AUTO-ICSI), whereas the remaining 89 participated in oocyte donor ICSI (DONOR-ICSI). Ejaculates were collected and DGC treated with and without MACS. Live birth and miscarriage rates resulting from ICSI observed after clinical pregnancy were determined. Sperm selection using DGC or a combination of DGC and MACS did not show any statistical difference with respect to live birth rate of couples undergoing either AUTO-ICSI or DONOR-ICSI, irrespective of whether the couples had a moderate (≥30 to <50%) or high (≥50%) level of SDF. Remarkably, there was no evidence of miscarriage in either cohort of patients (AUTO-ICSI or DONOR-ICSI) following the MACS procedure.

Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms.

Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection.

Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting.

ObjectiveThis study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency.MethodsSemen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated.ResultsThe sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%).ConclusionThe combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

BackgroundThe plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.ResultsWe show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.ConclusionA combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.

Differential expression of HNF4α isoforms in liver stem cells and hepatocytes

The hepatocyte nuclear factor (HNF4alpha) has been implicated in liver development and hepatocellular differentiation. The HNF4alpha expression in liver stem cells has not yet been fully investigated. Here, we describe the expression characterization of HNF4alpha in liver stem cells isolated from 2-acetylaminofluorene/partial hepatectomy (AAF/PH) rat model by a combination of density-gradient centrifugation and selective enzymatic digestion. The obtained cells were identified by flow cytometry (FCM) immediately and then by immunocytochemisty and reverse transcription-polymerase chain reaction (RT-PCR) after 5 d culture. The cultured stem cells were subjected to analysis of the expression of various HNF4alpha isoforms using RT-PCR, Western blotting, and electrophoretic mobility shift assay (EMSA). Results showed that HNF4alpha isoforms, alpha1, alpha2, alpha7, and alpha8 were present in liver stem cells, which contrasted with only alpha1 and alpha2 expression in hepatocytes. The distinct expression patterns of HNF4alpha isoforms in liver stem cells may contribute to maintain the "stemness" of these cells.

Advantage of combining magnetic cell separation with sperm preparation techniques

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.

PACAP Regulation of Secretion and Proliferation of Pure Populations of Gastric ECL Cells

The gastric enterochromaffin-like (ECL) cell plays a major role in the regulation of gastric acid secretion. We have previously described that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is present on myenteric neurons in the rat and colocalizes with its high-affinity receptor, PAC1, expressed on the surface of gastric ECL cells. The study of ECL cell physiology has been hampered by the inability to isolate and purify ECL cells to homogeneity. Density gradient elutriation alone yields only 65-70% purity of ECL cells. In the present study, we used fluorescence-activated cell sorting (FACS) with a novel fluorescent ligand, Fluor-PACAP-38, for isolating pure ECL cells. FACS was used to isolate ECL cells based on their relatively small size, low density, and ability to bind the fluorescent ligand Fluor-PACAP-38. The sorted cells were unambiguously identified as ECL cells by immunohistochemical analysis using anti-PACAP type-I (PAC1), anti-histidine decarboxylase (HDC), and anti-somatostatin antibodies. Further confocal microscopy demonstrated that Fluor-PACAP-38, a ligand with a higher affinity for PAC1, bound to extracellular receptors of these FACS-purified cells. FACS yielded an average of 2 million ECL cells/4 rat stomachs, and >99% of the sorted cells were positive for PAC1 receptor and HDC expression. The absence of immunohistochemical staining for somatostatin indicated lack of contamination by gastric D cells, which are similar in size and shape to the ECL cells. Internalization of PACAP receptors and a rapid Ca2+ response in purified ECL cells were observed upon PACAP activation, suggesting that these cells are viable and biologically active. These ECL cells demonstrated a dose-dependent stimulation of proliferation in response to PACAP, with a maximum of 30% proliferation at a concentration of 10-7 M. Microarray studies were perfor med to confirm the expression of genes specific for ECL cells. These results demonstrate that rat gastric ECL cells can be isolated to homogeneity by using a combination of density gradient centrifugation, followed by cell sorting using Fluor-PACAP. These techniques now allow microarray studies to be performed in ECL cells to characterize their functional gene expression and will facilitate pharmacological, biochemical, and molecular studies on ECL cell function.

Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.

Effect of Neutral Pyridine Leaving Groups on the Mechanisms of Influenza Type A Viral Sialidase-Catalyzed and Spontaneous Hydrolysis Reactions of α-d-N-Acetylneuraminides

A reagent panel, comprised of five pyridinium salts of α-d-N-acetylneuraminic acid, was synthesized and then used to probe enzymatic (α-sialidase) and nonenzymatic mechanisms of neuraminide hydrolysis. Spontaneous hydrolysis of the pyridinium salts proceeded via two independent pathways, where unassisted C−N bond cleavage was the rate-determining step. Cationic species (i.e., anomeric carboxylate protonated) displayed apparent pKa values in the range of 0.4−0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar βlg values of −1.22 ± 0.16 and −1.22 ± 0.07, respectively. The results, plus the activation parameters calculated from the hydrolysis of pyridinium α-d-N-acetylneuraminide (ΔH⧧ = 112 ± 2 kJ mol-1 and ΔS⧧ = 28 ± 4 J mol-1 K-1), strongly suggest that the anomeric carboxylate does not assist in the departure of neutral pyridine leaving groups. Enzymatic hydrolysis was studied using an influenza viral α-sialidase (A/Tokyo/3/67) which was recombinantly expressed using a baculovirus/insect cell expression system. Sialidase protein was purified by a combination of density gradient centrifugation and gel filtration chromatography. Kinetic parameters for the enzymatic hydrolysis of the pyridinium salts were measured at 37 °C and at pH values of 6.0 and 9.5. The βlg values derived for kcat/Km and kcat were essentially zero, indicating that chemical transformations/events are not rate-determining. Rather, this observation is consistent with a model for α-sialidase-catalyzed hydrolyses (Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L. J. Am. Chem. Soc. 1994, 116, 5572) in which kcat/Km is determined by a conformational change of the first-formed Michaelis complex and kcat is determined by the virtual transition state made up of two separate conformational events.

Purification of ovine somatotrophs using a combination of density gradient centrifugation and short-term culture

Freshly dispersed or cultured (18 h) ovine anterior pituitary cells were fractionated on 55% sigmoidal Percoll gradients. This resulted in the separation of two sub-populations of cells at densities of 1.075 g/ml (peak I) and 1.055 g/ml (peak II). Radioimmunoassay of fractions from a gradient on which cells were separated before culture showed the profiles of GH and prolactin to be virtually superimposable. After culture, however, the lactotroph population exhibited an apparent shift in its density profile, the lighter population increasing at the expense of the denser one. Immunocytochemistry of the cells remaining in the denser peak (peak I) showed that it consisted of about 65% somatotrophs which was approximately a three-fold enrichment over the proportion of somatotrophs present in the original cell preparation (24%). Refractionation studies indicated that the change in the density profile of the lactotrophs was due to an actual reduction in their density rather than a loss of viability of the denser sub-population. Secretion data showed the purified somatotrophs to be more responsive than the crude cell preparation to the regulatory factors, GH-releasing hormone and somatostatin. This enriched cell population should prove useful in the detailed study of GH secretion.

Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis.

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips.

The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11-45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.