Antigen Presentation and Proteome Study of Exosomes Secreted by Co-Culture of Macrophages and Talaromyces marneffei.

It is known that intracellular pathogens interact and react with the cellular immune system through exosomes produced by macrophages. This study aimed to determine whether co-culture of macrophages and Talaromyces marneffei induces exosomes and leads to immune responses. T. marneffei was incubated to collect conidia, co-cultured with human macrophages, which then induced exosomes. In cellular experiments, after extraction and purification, the exosomes were then observed by electron microscopy and detected by flow cytometry and mass spectrometry. In animal experiments, flow cytometry and enzyme-linked immunosorbent assay were used to examine whether exosomes were antigenpresenting. The results showed that purified exosomes produced a pro-inflammatory response and stimulated production of TNF-α in non-fungal-treated macrophages. Protein mass spectrometry analysis of exosomes also indicated their potential ability to activate the internal immune response system and the pro-inflammatory response. Translation and ribosomes were the most abundant GO terms in proteins, and the most relevant KEGG pathway was the biosynthesis of secondary metabolites. Furthermore, in vivo experiments revealed that exosomes induced activation of lymphocytes and increased expression of TNF-α and IL-12 in the lung, mediastinum, and spleen area. In conclusion, exosomes can be released by co-culture of T. marneffei and macrophages, having antigen-presenting functions, promoting macrophage inflammation, and initiating adaptive immune responses. These processes are inextricably linked to the translation of secondary metabolites, ribosomes and biosynthesis.

[Cross-reaction of seasonal influenza vaccine immune serum against Eurasian Avian-like H1N1 swine influenza virus].

Objective: To evaluate the cross-reaction of seasonal influenza vaccine immune serum against Eurasian avian-like H1N1 swine influenza virus. Methods: Nine human infected Eurasian avian-like H1N1 swine influenza virus strains were obtained from national influenza surveillance network laboratories in Jiangsu, Hebei, Shandong, Yunnan, Hunan, Fujian and Tianjin provinces, and their genetic characteristics of hemagglutinin were analyzed by deep sequencing. 30 volunteers were recruited respectively from children (2-5 years old), adults (24-57 years old) and elderly (60-84 years old) who received 2019-2020 seasonal influenza vaccine in Anning city, Yunnan Province in October 2019, and serum samples were collected before and 1 month after vaccination. The hemagglutination inhibition test was used to evaluate the cross-reaction of serum before and after immunization against 4 strains of human infection with Eurasian avian-like H1N1 swine influenza virus isolated since 2015. Results: The homology of hemagglutinin genes of 9 Eurasian avian-like H1N1 swine influenza viruses was similar, but the difference of hemagglutinin heavy chain and light chain amino acid genes with A (H1N1) pdm09 (vaccine strain) were 90-101 and 24-30 amino acids respectively. The antibody titer of vaccine strain antiserum to vaccine strain was 2 560; the antibody titers of the vaccine strain antiserum to Eurasian avian-like H1N1 swine influenza virus and the Eurasian avian-like H1N1 swine influenza virus antiserum to vaccine strain were same as 640. The proportion of children, adults and elderly vaccinated with seasonal influenza vaccine with antibody titer ≥40 against vaccine strain was 90.0%, 70.0% and 73.3%, respectively; while the proportion merely were 46.7%, 36.7% and 33.3%-43.3% to 4 strains of Eurasian avian-like H1N1 swine influenza virus, respectively. Conclusion: Seasonal influenza vaccination does not provide effective cross-protection against Eurasian avian-like H1N1 swine influenza virus.

Regulatory effect of miRNA on multi-directional differentiation ability of mesenchymal stem cell in treatment of osteoporosis.

This study was designed to evaluate the effect of miRNA acting in regulating multi-directional differentiation ability of mesenchymal stem cell in treatment of osteoporosis (OP), with the aim of finding a new idea and approach for clinical treatment of OP. Estrogen deficiency-induced OP mice model was established by means of ovariectomy (OVX). Additionally, a sham group was set up for control. Bone Marrow Mesenchymal Stem Cells (BMMSCs) of OVX group (O/BMMSCs) and BMMSCs of sham group (S/BMMSCs) were separately cultured. Then surface markers of BMMSCs were detected. Multi-directional differentiation ability was identified in the two groups by giving cells targeted induced stimulation. It was found that the bone trabecula, bone density and bone volume fraction of distal femoral metaphysis in the OVX group were much lower than those of the sham group. Moreover, trabecular bone space in the OVX group became larger; O/BMMSCs and S/BMMSCs both had normal expression of surface markers as well as potentials of osteogenic and adipogenic differentiation; O/BMMSCs had a weaker osteogenic capability but a stronger adipogenic capability than S/BMMSCs. All the findings suggest that the regulatory effect of miRNA on multi-directional differentiation ability plays a vital role in the treatment of OP, and there is a close correlation between them; deficiency or functional defect of BMMSCs can result in the occurrence of OP.

[Relationship between smoking and the risk of morbidity of chronic obstructive pulmonary diseases among residents aged 30 years and above in Sichuan Province: a prospective study].

Objective: To investigate the morbidity of chronic obstructive pulmonary diseases (COPD) in residents aged 30 years and above in Sichuan Province, and analyze the effect of smoking on the risk of morbidity on COPD. Methods: From 2004 to 2008, people were randomly selected from Pengzhou, Sichuan Province. All the local people aged 30-79 years were asked to receive questionnaire survey, physical examination and pulmonary function testing, and long-term follow-up to determine the morbidity of COPD. Cox proportional hazard regression model was used to analyze the relationship between smoking and COPD. Results: In 46 540 participants, the current smoking rates were 67.31% in males and 8.67% in females, there were 3 101 new cases of COPD, with a cumulative incidence of 6.66%. Adjusted for age, gender, occupation, marriage, income level, educational level, BMI, daily total physical activity, current cooking frequency, whether there was smoke exhaust device at present and frequency of passive smoking exposure, multivariate Cox proportional hazard regression analysis showed that compared with the non-smoking population, current smoking and quitting smoking increased the risk of COPD, with HR of 1.42 (95%CI:1.29-1.57) and 1.34 (95%CI:1.16-1.53). Compared with people who never or occasionally smoke, the risk of morbidity on COPD increased with the increase of average daily smoking volume, mixed smoking at present, mixed smoking at the beginning increased the risk of COPD, with HR of 1.79 (95%CI: 1.42-2.25) and 2.12 (95%CI: 1.53-2.92), started smoking at the age of <18 years old and ≥18 years old increased the risk of COPD, with HR of 1.61 (95%CI:1.43-1.82) and 1.34 (95%CI: 1.22-1.48), inhaling into the mouth, throat and lung during smoking increased the risk of COPD, with HR of 1.30 (95%CI: 1.16-1.45), 1.63 (95%CI: 1.45-1.83) and 1.37 (95%CI: 1.21-1.55). Adjusted for multiple confounding factors and adjusted for regression dilution bias, the average daily smoking volume, the age of starting smoking and the depth of smoking inhalation had an impact on the incidence of COPD, and the gender difference was particularly prominent. Conclusions: Smoking increased the risk of morbidity on COPD, which was related to the average daily smoking volume, the type of smoking, the age of starting smoking and the depth of smoking inhalation. Tobacco control should comprehensively consider the specific characteristics of smoking, so as to prevent COPD.

The involvement of senescence induced by the telomere shortness in the decline of osteogenic differentiation in BMSCs.

OBJECTIVE: BMSC (Bone marrow mesenchymal stem cells) is an important seed cell for the repair of bone and cartilage defect in the tissue engineering. The proliferation rate and differentiation capacity of BMSCs from the old donors were less than that from young donors; however, the related mechanism remained unclear. MATERIALS AND METHODS: Sprague Dawley (SD) rats BMSCs were cultured and treated. Hydrogen peroxide (H2O2) and continual passage of BMSCs were performed to induce senescence. Senescence was detected by the SA-b-Gal staining and the telomere length analysis. Cell proliferation and osteogenic differentiation were also observed. Finally, Olaparib was used to maintain the telomere length and investigate the role of telomere length and senescence on the cell proliferation and differentiation. RESULTS: H2O2 could increase the positive rate of SA-b-Gal staining in BMSCs and shorten the length of the telomere. The proliferation rate and ALP activity were also decreased by the H2O2. The senescence and decline of osteogenic differentiation could also be observed after prolonged passage of BMSCs. Inhibition of telomere length decline could attenuate the increased positive rate of SA-b-Gal staining induced by the H2O2, promote the cell proliferation, and enhance the capacity of osteogenic differentiation. CONCLUSIONS: Senescence induced by the decline of telomere length could reduce the capacity of osteogenic differentiation and inhibit cell proliferation in BMSCs.

Influence of VEGF/BMP-2 on the proliferation and osteogenetic differentiation of rat bone mesenchymal stem cells on PLGA/gelatin composite scaffold.

OBJECTIVE: To investigate the influence of VEGF/BMP-2 on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells BMSCs) on PLGA/gelatin composite scaffold. MATERIALS AND METHODS: Randomly-oriented nanofibers with different ratios of Poly Lactic-co-Glycolic Acid (PLGA)/gelatin were produced through electrospinning. The mixture of nanofibers and BMSCs was pipetted onto the surface of the scaffolds, and BMSCs/PLGA/gelatin composite was obtained. The surface morphology, chemical structure, hydrophilicity and mechanical property of PLGA/gelatin nanofibers were revealed by scanning electron microscope. In vitro release kinetics of bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) were studied using ELISA kits. The cell adhesion, growth and proliferation of BMSCs on scaffolds were observed by scanning electron microscopy. The CCK-8 assay was used to evaluate the effects of VEGF/BMP-2 slow release system on the proliferation of BMSCs on scaffolds. RT-PCR was used to examine the activities of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX-2), and osteocalcin (OCN). RESULTS: In each group of cells in the in-vitro experiment, through electron microscope scanning, fiber scaffolds were interconnected three-dimensional reticular structure, BMSCs firmly attached to the fiber surface and internal stent, cells experienced a long spindle, polygon change, and branch-like protrusions on the cell surface were connected. Under the electron microscope, cell proliferation curve and osteogenesis markers (ALP, RUNX-2, OCN) expression in the dual factor group on cell adhesion, proliferation and differentiation were much better than those of blank control group and single factor groups. CONCLUSIONS: In the successfully constructed gelatin/PLGA nanofiber scaffold, VEGF and BMP-2 can be sequentially released, during which VEGF and BMP-2 can promote the adhesion, proliferation, and differentiation of BMSCs.

[Association of RTN4 gene rs2864052 and rs6545468 with the susceptibility of nasopharyngeal carcinoma in Guangxi Zhuang population].

Objective:To study the relationship of the polymorphism of RTN4 gene rs2864052 and rs6545468 and haplotype with the susceptibility of nasopharyngeal carcinoma in Guangxi Zhuang population. Method:The polymorphism of Nogo gene (rs2864052,rs6545468) and haplotype were analyzed using the method of single-base extension PCR and DNA sequencing in 282 cases of nasopharyngeal carcinoma (NPC) and 199 healthy persons (control group) in Guangxi Zhuang Autonomous Region. Result:There were no differences between the NPC's patients and controls in the genotype and allele frequencies of RTN4 gene rs2864052 site,or rs6545468 site. The frequency of AG haplotype in the NPC's patients was significantly lower than in the controls(P=0.004, OR=0.14,95%CI=0.31-0.68). Conclusion:The haplotype AG of RTN4 gene rs2864052 and rs6545468 sites may reduce the risk of nasopharyngeal carcinoma in Guangxi Zhuang population.

Effect of chymosin action on the hydrodynamic diameter of casein micelles.

Quasi-elastic light scattering shows an initial decrease of about 5 nm in the hydrodynamic radius of casein micelles after adding chymosin, assuming the decrease to be equal for all micelles. This is consistent with the hypothesis that casein micelles have a hairy outer layer that is partly made up of the caseino-macropeptide part of kappa casein.

Variation of craniocervical junction volume as an effective parameter for basilar invagination treatment.

OBJECTIVE: The major pathological change in basilar invagination (BI) is represented in the decrease of craniocervical junction (CVJ) volume resulting from abnormal bone protrusion around the foramen magnum. The diagnosis and clinical evaluation of BI is mainly based on the clinical manifestations and radiographic measurements by means of calculation of the scan lines of CVJ in X-ray, CT and MRI. With the transoral decompression atlantoaxial reduction plate (TARP III) system, the decompression, reduction and fixation can be achieved to decompress and stabilize medulla spinalis change the position of the dens in CVJ, thus expand the CVJ relative volume, relieve the compression on medulla spinalis and the nerve injury. However, the correlation between the dens position change and the variation of CVJ has not been established previously. This study focused on the clinical significance of the variation of craniocervical junction (CVJ) volume caused by the dens position change for the treatment of BI. PATIENTS AND METHODS: We've performed an analysis of data from 62 BI patients admitted from January 2008 to May 2013, who were treated by TARP III system. The data include preoperative, postoperative JOA scores (Japanese Orthopaedic Association scores, 17 points method), preoperative and postoperative X-ray, thin-slice CT scan with three-dimensional reconstruction and MRI scan to measure the cervicomedullary angle (CMA). We have analyzed the preoperative and postoperative three-dimensional CT data by means of MIMICS 10.01 software system according to the Box volume (BV) method to determine the changes of CVJ volume resulting from preoperative and postoperative dens position change, assessed the correlation between the CVJ volume changes and the JOA scores with correlation between CMA change and the JOA scores. All data were analyzed by paired t-test and Pearson correlation analysis. RESULTS: In all 62 patients, JOA scores were recovered from preoperative 9.26 ± 1.66 to postoperative 13.02 ± 1.44, CMA change rate was 21%, and CVJ volume change rate was 36%. The CMA change rate and the JOA score recovery rat exhibited relevance, as Pearson's correlation coefficient was 0.46 (p < 0.005). The Pearson's correlation coefficient between CVJ volume change rate and JOA score recovery rate was 0.63 (p < 0.005), and the CVJ volume change rate was significantly different while compared with the correlation between CMA change rate and JOA score (p < 0.005). CONCLUSIONS: the CVJ volume change rate is a sensitive and reliable parameter for the evaluation of neurological function improvement in patients with BI. It can be used as a predictor to evaluate the postoperative neurological recovery.

High-energy collision-induced dissociation of ceramide ions from permethylated glycosphingolipids.

Ceramide fragments from permethylated glycosphingolipids (GSLs) were studied by highenergy collision-induced dissociation (CID). In comparison with ceramlde fragments of underivatized GSLs, many more product ions including charge-remote fragment ions were observed. These ions provided detailed structural information on the ceramides. The relative intensity and the mass interval between the L and M ions were used to assign the position of the double bond. The position of the hydroxyl group was assigned with the Ln and K ions. Because the ceramide fragments and not the pseudomolecular ions were selected as the precursor ions, the size of GSLs had little effect on the quality of the product ion spectra. The sensitivity of this approach was in the range of picomoles.

Determination of polymer size distribution by combination of quasielastic light scattering and band transport: evaluation of the effect of diffusion.

In this paper we report a computer simulation study of the effect of diffusion on the size distribution obtained by combining light scattering with isokinetic band sedimentation or electrophoresis. We find that, under typical experimental conditions, the method yields reasonably accurate size distributions for samples of particles greater than 10 nm radius. However, caution should be exercised in interpreting the results for smaller particles, for which the distortion due to diffusion can be considerable.

Chemistry and antioxidative factors in rosemary and sage.

Rosemary and sage are common spices used in food. In our recent search of cancer chemopreventive agents from spices, the alcohol extracts of rosemary and sage showed strong antumorigenic activities. Rosemary and sage extracts contain active antioxidative factors such as phenolic diterpenes, flavonoids and phenolic acids. Here we discuss chromatographic methods used to separate and purify compounds from these spices and MS and NMR spectrometry to identify the isolated compounds. Several new compounds isolated from sage were determined to be 6-O-caffeoyl-beta-D-fructofuranosyl-(2-->1)-beta-glucopyranoside, 1-O-caffeoyl-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, 1-O-p-hydroxybenzoyl-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, 1-O-(3-methyl-2,3,4-trihydroxybutyl)-6-O-feruloyl-beta-D-glucopyranoside, 4-hydroxyacetophenone 4-O-[5-O-(3,5-dimethoxy-4-hydroxybenzoyl)-beta-D-apiofrunosyl]-(1-->2)-beta-D-glucopyranoside and 1-O-[2-hydroxy-5-(2-hydroxyethyl)phenyl]-6-O-trans-caffeoyl-beta-D-glucopyranoside.

Trypanosoma congolense infections: MHC class II-restricted immune responses mediate either protection or disease, depending on IL-10 function.

BALB/c mice are highly susceptible and C57BL/6 relatively resistant to Trypanosoma congolense infections. Here we show that relatively resistant wild-type B6 mice infected with T. congolense survive significantly longer (> 200 days) than infected major histocompatibility complex (MHC) class II-deficient B6 mice (approximately 50 days). We also show that blocking of the interleukin-10 (IL-10) receptor induces early death of wild-type B6 mice infected with T. congolense (approximately 10 days), but does not affect the survival of infected MHC class II-deficient B6 mice. We conclude that MHC class II-restricted immune responses mediate protection and, when IL-10 function is impaired, MHC class II-restricted immune responses mediate early mortality in otherwise resistant B6 mice. Thus, in T. congolense infections, MHC class II-restricted immune responses mediate either protection or disease, depending on IL-10 function.

Experimental African trypanosomiasis: lack of effective CD1d-restricted antigen presentation.

BALB/c mice are highly susceptible to African trypanosomiasis, whereas C57BL/6 mice are relatively resistant. Other investigators have reported that the synthesis of IgG antibodies to purified membrane form of variant surface glycoprotein (mfVSG) of Trypanosoma brucei is CD1 restricted. In this study, we examine the role of the CD1d/NKT cell pathway in susceptibility and resistance of mice to infection by African trypanosomes. Administration of anti-CD1d antibodies to Trypanosoma congolense-infected BALB/c mice neither affects the parasitemia nor the survival time. Correspondingly, CD1d(-/-) and CD1d(+/+) BALB/c mice infected with T. congolense or T. brucei show no differences in either parasitaemia or survival time. The course of disease in relative resistant C57BL/6 mice infected with T. congolense is also not affected by the absence of CD1d. Parasitaemia, survival time, and plasma levels of IgG2a and IgG3 parasite-specific antibodies in infected CD1d(-/-) C57BL/6 are not different from those of infected CD1d(+/+) C57BL/6 mice. We conclude that CD1d-restricted immune responses do not play an important role in susceptibility/resistance of mice infected with virulent African trypanosomes. We speculate that virulent trypanosomes have an evasion mechanism that prevents the induction of a parasite-specific, CD1d-restricted immune response by the host.

High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.

Stopped-flow studies of myelin basic protein association with phospholipid vesicles and subsequent vesicle aggregation.

When mixed with vesicles containing acidic phospholipids, myelin basic protein causes vesicle aggregation. The kinetics of this vesicle cross-linking by myelin basic protein was investigated by using stopped-flow light scattering. The process was highly cooperative, requiring about 20 protein molecules per vesicle to produce a measurable aggregation rate and about 35 protein molecules per vesicle to produce the maximum rate. The maximum aggregation rate constant approached the theoretical vesicle-vesicle collisional rate constant. Vesicle aggregation was second order in vesicle concentration and was much slower than protein-vesicle interaction. The highest myelin basic protein concentration used here did not inhibit vesicle aggregation, indicating that vesicle cross-linking occurred through protein-protein interactions. In contrast, poly(L-lysine)-induced vesicle aggregation was easily inhibited by increasing peptide concentrations, indicating that it did cross-link vesicles as a peptide monomer. The myelin basic protein:vesicle stoichiometry required for aggregation and the low affinity for protein dimerization suggested that multiple protein cross-links were needed to form a stable aggregate. Stopped-flow fluorescence was used to estimate the kinetics of myelin basic protein-vesicle binding. The half-times obtained suggested a rate constant that approached the theoretical protein-vesicle collisional rate constant.

Calcium effects on prothrombin and its reaction with bifunctional alkylating reagents.

Monodisperse bovine prothrombin was prepared and its molecular states under several conditions examined. The protein showed no tendency to self-associate in the absence of calcium. Calcium (4 mM) caused small increases in the apparent molecular weight of the protein which may or may not represent protein dimerization with very low affinity. The allowed conclusion was that calcium-induced prothrombin dimerization is minimal up to protein concentrations of many mg/ml. Calcium-induced protein shape changes did not measurably alter the protein diffusion constant. A bifunctional alkylating reagent did produce extensive calcium-dependent prothrombin crosslinking. Prothrombin dimers formed by the crosslinking agent were not a measure of the state of native prothrombin.

Electron microscopy and hydrodynamic properties of blood clotting factor V and activation fragments of factor V with phospholipid vesicles.

The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.

Kinetic and hydrodynamic analysis of blood clotting factor V-membrane binding.

The kinetics and hydrodynamic properties of factor V-membrane interaction were characterized. Factor V bound to membranes containing acidic phospholipids with a high collisional efficiency. For membranes of 20% phosphatidyl-serine-80% phosphatidylcholine, an association rate constant of (1.13 +/- 0.10) X 10(8) M-1 s-1 was obtained. These membranes contained about 20 factor V binding sites per vesicle of 3.6 X 10(6) daltons. This association rate represented about a 30% collisional efficiency. Dissociation of factor V was measured by a fluorescence energy transfer method with a dissociation rate constant of 0.0055 s-1 at 10 degrees C. The equilibrium dissociation constant for binding to these membranes at 10 degrees C and 0.14 M ionic strength was 5 X 10(-11) M. Ionic strength, pH, calcium, and charge density in the membrane had large effects on the rate of factor V-membrane dissociation, indicating a strongly ionic interaction between protein and membrane. In contrast, the association rate was nearly insensitive to ionic strength. The membrane-binding properties were relatively unchanged after thrombin digestion of factor V or after long-term protein storage which resulted in loss of procoagulant activity. Other proteins of the prothrombinase reaction greatly decreased the rate of factor Va-membrane dissociation. At protein saturation, factor V increased the hydrodynamic radius of phospholipid vesicles by 11.4 nm. In contrast, factor Va increased the hydrodynamic vesicle radius by only about 5 nm. The mass of membrane-bound protein was comparable for both proteins.

Deglycosylated prothrombin fragment 1. Calcium binding, phospholipid interaction, and self-assocation.

The carbohydrate portion of prothrombin fragment 1 has been removed by fluorolysis in anhydrous HF. The deglycosylated protein retains its calcium- and membrane-binding properties. The slow, calcium-dependent protein transition monitored by changes in intrinsic protein fluorescence remains intact for the aglycoprotein. Calcium-dependent protein-membrane binding is also observed and can be quantitatively reversed with EDTA. The major alteration resulting from carbohydrate removal is the degree of protein self-association. Both the normal and deglycosylated proteins undergo a rapid self-association which approaches a dimer in the presence of calcium. This self-association is independent of the slow change in intrinsic fluorescence. The deglycosylated protein then undergoes a secondary self-association with kinetics identical with the fluorescence change. This secondary self-association also occurs on the membrane surface. This suggests that the calcium-dependent conformational change exposes a site on the protein which functions in secondary self-association. The carbohydrate apparently masks this site in the native molecule.