Molecular Ultrafiltration Research Articles

A Novel Cryptococcal Meningitis Therapy: The Combination of Amphotericin B and Posaconazole Promotes the Distribution of Amphotericin B in the Brain Tissue.

The deficient brain tissue distribution of amphotericin B (AMPB) seriously restricts its treatment for the clinical efficacy of cryptococcus neoformans meningitis (CNM). We strive to develop a tactic to increase its concentration in brain tissue. We aimed to investigate whether the combination of AMPB and posaconazole (POS) could be more effective in the treatment of CNM and to elucidate its potential mechanisms. HPLC analysis was used to analyze the concentration of AMPB in mouse serum, brain tissue, and BCECs cells. Schrodinger molecular docking, in vitro plasma balance dialysis, and ultrafiltration analysis were performed to evaluate the combinative effect of AMPB and POS with serum albumin and POS on AMPB plasma protein binding. H&E staining and colonization culture experiment of CN were employed to assess the effect of POS on the efficacy of AMPB. POS + AMPB significantly reduced the concentration of plasma total AMPB and increased its concentration in the brain tissue. However, the P-gp inhibitor zosuquidar, BCRP inhibitor Ko143, and a common inhibitor of both, elacridar, had no significant effect on its concentration. Molecular docking, balance dialysis, and ultrafiltration analysis showed that AMPB and POS had potential binding properties to serum albumin. Meanwhile, 4 and 8 μg/mL POS could significantly increase the concentration of free AMPB in plasma. POS and three inhibitors all had no significant effect on the uptake of AMPB by BCECs, but serum albumin had. The therapeutic effect of CNM in mice was confirmed that AMPB and AMPB+POS could restrain the infiltration of neutrophils and lymphocytes in cortical neurons and improve the bleeding and markedly inhibit the proliferation of CN. Collectively, we propose that POS competitively binds to the plasma protein sites of AMPB, thereby increasing its level in the brain tissue. Meanwhile, POS could enhance the efficacy of AMPB in the treatment of CNM, which may be independent of P-gp and BCRP proteins.

Lysyl Oxidase-like-2 Cross-links Collagen IV of Glomerular Basement Membrane

The 7S dodecamer is recognized as an important structural cross-linking domain of collagen IV networks that provide mechanical stability to basement membranes, a specialized form of extracellular matrix essential for the development and maintenance of tissue architecture. Although the 7S dodecamer is stabilized by covalent cross-linking, the molecular mechanism by which such cross-links are formed has not been revealed. Here, we aimed to identify the enzyme(s) that cross-links the 7S dodecamer and characterize its expression in the kidney glomerulus. Pharmacological inhibition of candidate extracellular matrix enzymes revealed that lysyl oxidase activity is required for cross-linking of 7S polypeptides. Among all lysyl oxidase family members, lysyl oxidase-like-2 (LOXL2) was identified as the isoform cross-linking collagen IV in mouse embryonal PFHR-9 cells. Biochemical analyses revealed that LOXL2 readily promoted the formation of lysyl-derived cross-links in the 7S dodecamer but not in the NC1 domain. We also established that LOXL2 is the main lysyl oxidase family member present in the glomerular extracellular matrix. Altogether, we demonstrate that LOXL2 is a novel component of the molecular machinery that forms cross-linked collagen IV networks, which are essential for glomerular basement membrane stability and molecular ultrafiltration function.

Construction and Characterization of B850-Only LH2 Energy Transfer System in Purple Bacteria

To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis, the conditions for construction of B850-only peripheral light-harvesting complex (LH2) and their properties were investigated using absorption, fluorescence spectroscopy, molecular sieve chromatography, ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the purple bacteria. The results indicated that bacteriochlorophylls (BChl) of B800 incubated in 10 mmo · L(-1) Tris-HCl (pH 8.0) buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans (A-LH2) by 0.08% (W/V) SDS. B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration. B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature (RT). While BChl of B800 incubated in pH 1.9 buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris (P-LH2). The authors acquired two components using molecular sieve chromatography. Free BChl of one component was not removed and self-assembled to P-LH2. The other removed free BChl and B850-only P-LH2 was constructed. B850 unchanged after P-LH2 was incubated. P-LH2 α and β subunits have different molecular weights, but those of A-LH2 are in the contrary. It is concluded that B850-only P-LH2 is more stable than A-LH2. The enigmatic split of the B800 absorption band was not observed in these LH2, but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris. The authors' results may provide a new light to separate homogeneous Apoprotein LH2.

Polyvalent horse F(Ab`) 2 snake antivenom: Development of process to produce polyvalent horse F(Ab`) 2 antibodies anti-african snake venom

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica crude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and the immunoglobulins were separated from the sera by (NH4)2SO4 precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22
m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed by the ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethality of venoms, as estimated by ED50 determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Antigenic specificity of human serum γ-globulin samples obtained under conditions of equimolar binding of copper and zinc cations

Samples of human serum gamma-globulin modified by equimolar binding of copper and zinc cations were obtained using the method of molecular ultrafiltration. Conformation characteristics of the protein were studied by UV spectrophotometry. Immunochemical study included radial immunodiffusion test, direct and sandwich enzyme immunoassays. Conformation changes in gamma-globulin caused by incorporation of solitary metal cations into the protein molecule modified presentation of antigenic determinants on the globule surface and their availability for recognition by specific antibodies. New antigenic determinants and new antigenic specificity of gamma-globulin are not formed under these conditions.

Preparation of specimens of human serum γ-globulin modified by copper and zinc and its immunochemical characteristics

Specimens of human serum gamma-globulin modified by molar excess of copper and zinc cations were obtained by molecular ultrafiltration. Conformation characteristics of the protein were determined by UV spectrophotometry. Immunochemical study included radial immunodiffusion test and direct and sandwich enzyme-linked immunosorbent assay. After binding of copper and zinc, the gamma-globulin molecule underwent conformation changes modifying presentation of antigenic determinants on the globule surface and their availability for recognition by specific antibodies. The effects of copper were much more pronounced than those of zinc cations.

Binding of zinc cations to human serum γ-globulin

Binding of zinc cations to human serum gamma-globulin was studied by molecular ultrafiltration. The content of free metal in the filtrate was evaluated by reaction with o-phenanthroline. Conformation characteristics of the protein were determined by UV spectrophotometry. Our findings suggest that gamma-globulin molecule contains several zinc binding sites differing by corresponding constants and successively occupied with increase in the content of bound metal. The parameters of zinc binding correspond to those obtained in experiments with copper. Conformation status of protein with bound zinc differs significantly from that of protein with bound copper cations.

Human serum γ-globulin binds copper cations

Binding of copper cations to human serum gamma-globulin was studied using molecular ultrafiltration. The content of free metal in the filtrate was evaluated by the reaction with sodium diethyldithiocarbamate. Conformation characteristics of the protein were evaluated by UV spectrophotometry. gamma-Globulin molecule has several copper-binding sites differing by binding constants and filled one-by-one as the content of bound metal increased.

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF.

Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.

Crystal Structure of NC1 Domains: STRUCTURAL BASIS FOR TYPE IV COLLAGEN ASSEMBLY IN BASEMENT MEMBRANES

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.

Characterization of a protein recognizing minor groove binders-damaged DNA.

By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.

Characterization of stable high molecular weight mutagenic product(s) of plant-activated m-phenylenediamine

Monocyclic aromatic amines are environmental contaminants and many are promutagens and procarcinogens. Cultured tobacco cells, strain TX1, activated m-phenylenediamine into a frameshift mutagen that reverted the hisD3052 allele in Salmonella typhimurium strains TA98 and YG1024. However, the plant-activated products were refractory in strain TA98/1,8-DNP 6. This indicated that these plant-activated products were substrates for bacterial acetyl-CoA: N-hydroxyarylamine O-acetyltransferase. A stable, high molecular weight ( > 300 kDa) proximal mutagen was isolated by molecular ultrafiltration membranes. No parent compound was associated with the isolated mutagenic fraction. The high molecular weight fraction induced mutation in S. typhimurium strains TA98, YG1021 and YG1024. From these data we propose a model for the plant-activation of aromatic amine promutagens.

Validation of an ultrafiltration process to concentrate viruses from large volumes of water

Abstract The efficiency of seeded poliovirus and echovirus recovery from 20 litres of tap water was evaluated using a molecular ultrafiltration system. The experiments were performed using a 5μm polypropylene prefilter and two polysulfonate membranes of 105 nominal molecular weight limit. No loss of virus was observed after prefiltration. More than 96% of poliovirus were recovered after elution at pH 10.5 and 83% of echovirus after elution at pH 8.5.

Induction of transformational changes in normal endothelial cells by cultured human astrocytoma cells

Endothelial cell proliferation is a significant biological feature of malignant astrocytomas. The ability of the cells of these tumors to elaborate mitogenic angiogenesis factors has been well documented. However, less is known about the transformational effects that neoplastic astrocytes may have on the endothelial cells within malignant astrocytomas. In this study, the hypothesis that humoral factors elaborated by cells derived from malignant astrocytomas induce transformational changes in normal endothelial cells in vitro is investigated. Conditioned medium (CM) was prepared from exponentially growing cultures of a human glioblastoma cell line (UW18) and from two rat brain-tumor cell lines: an anaplastic astrocytoma (R175A) and a glioblastoma with sarcomatous elements (9L). Subconfluent target bovine aortic arch endothelial cells (BAEC's) were exposed for 48 hours to varying concentrations of CM prepared from each of these tumors, and then evaluated for transformational changes. Different molecular weight (MW) fractions of UW18 CM were prepared by molecular ultrafiltration, and each fraction was tested for transforming activity. Transformation endpoints included changes in cellular deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) content and distribution (measured by differential flow cytometry) and changes in de novo DNA synthesis determined by 3H-thymidine incorporation. Significant changes in the amount and distribution of DNA and RNA were observed in the BAEC's treated with UW18 CM compared to untreated BAEC's. At 10% concentrations of UW18 CM, changes in the RNA profile of target BAEC's were evident, and at 30% concentrations of UW18 CM, an irregular bimodal distribution was well established. Patterns of DNA were also altered in a concentration-dependent manner, with significant aneuploidy developing at UW18 CM concentrations of 20%. The DNA synthesis in BAEC's increased with increasing CM concentrations, up to a maximum of about 250% of control values at 30% concentrations of UW18 CM. The transformational changes induced after exposure of BAEC's to CM prepared from R175A and 9L were significantly less than those observed with UW18 CM. Molecular ultrafiltration was used to prepare UW18 CM fractions with MW cutoffs of less than 10 kD, 10 to 30 kD, and greater than 30 kD. Transformational activity was significant only in CM's with an MW of 10 to 30 kD. It is concluded that the UW18 human glioblastoma cell line elaborates a soluble factor, or group of factors, with an MW in the 10- to 30-kD range, capable of inducing transformational alterations in target normal endothelial cells, and that such transformation may account for some of the abnormal endothelial cell changes associated with malignant astrocytomas.

Isolation of wild-type hepatitis A virus from the environment

Three isolates of human hepatitis A virus (HAV) were cultivated directly from polluted Tiber water on Frp/3 cells. From 50 to 125 l. of water were prefiltered through 10 and 1 μm polygard filters and concentrated using a molecular ultrafiltration system. The prefilters were washed with 500 ml of beef-extract pH 9.0–9.5 to remove any adsorbed virus. The washing buffer was acid-pH precipitated or concentrated by ultrafiltration. Each sample was neutralized with a pool of antibodies against all the enteroviruses excluding hepatitis A virus. Three samples obtained from prefilters were scored as positive. Hepatitis A virus was identified by commercial ELISA test, indirect immunofluorescence, neutralization test, performed with polyclonal or monoclonal antibody, and by hybridization test with cDNA probes.

Determination of styrene and 2-vinylpyridine monomers in poly(2-vinylpyridine—styrene)

Residual monomers such as styrene can be determined in polymers at microgram per kilogram concentrations by headspace techniques. However, such techniques often do not have adequate sensitivity for the determination of monomers less volatile than styrene. A method is described for the determination of trace concentrations of monomers that are less volatile than styrene and also monomers that are traditionally determined in polymers by headspace techniques. The method involves dissolution of the polymer, removal of the polymer by molecular ultrafiltration and determination of the monomer in teh filtrate by gas chromatography—mass spectrometry. Concentrations of styrene and 2-vinlpyridine of less than 200 μg/kg were successfully determined in a copolymer used for the post-ruminal delivery of nutritional supplements and drugs.

Mutagen formation in a model beef boiling system I. Conditions with a soluble beef‐derived fraction

Abstract Fried beef and commercial beef extract contain Ames/ Salmonella frameshift mutagens that require microsomal (S‐9) activation. To ascertain which fraction(s) of beef muscle contain(s) the essential precursors, aqueous (1:1) homogenates of lean round steak were centrifuged to give an insoluble residue1 (R1) and a soluble supernatant1 (S1). S1 was then boiled for 30 min and again centrifuged, yielding a residue2 (R2) and a soluble supernatant2 (S2). S2 represents only 5% of the meat dry wt and it contains only 10∗ of the H2O‐soluble protein, but it contributes all of the S‐9 dependent Salmonella TA1538 mutagenic activity in boiled homogenates. Mutagen formation from S2 boiled for 0–30 h at a constant volume increases exponentially with time and displays sharp pH optima at 4.0 and 9.0. By molecular ultrafiltration the pH 4.0 mutagen precursors in S2 have molecular wts < 500. They are also stable to lyophilization. These observations and the disappearance of certain amino acids upon boiling at pH 4.0 ...

Lack of a direct nitrate-trifoliin A interaction in the Rhizobium-clover symbiosis

FRANK B. DAZZO and ESTELLE M. HRABAK Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 USA Received 22 July 1982 Key words Clover Combined nitrogen Lectin Nitrate Nitrogen-13 Rhizobium trifolii Symbiosis Trifoliin A Trifolium repens Summary Nitrate added at critical concentrations to plant growth medium inhibits the infection of legume roots by Rhizobium. The direct interaction of nitrate and trifoliin A, a Rhizobium-recognition lectin from white clover (Trifolium repens L.), was examined as a possible basis for this regulation. Selective molecular ultrafiltration studies to detect ligand-protein interactions showed that radioactive 13NO3- did not bind directly to trifoliin A when incubated at two molar ratios. Immunoprecipitation of trifoliin A by its homologous antibody was unaffected by 15 mM NO3 -. In addition, there was no apparent reduction in attachment of R. trifolii 0403 to root hairs of clover seedlings during l h of incubation in the presence of 15 mM NO 3 -. These results show that nitrate inhibition of these early steps of the infection process is not due to a direct interaction of nitrate with trifoliin A or its glycosylated receptors. Introduction Combined nitrogen is one of the many environmental factors which can suppress the development of the nitrogen-fixing Rhizobium-legume symbiosis. For instance, R. trifolii 0403 does not form root nodules on white clover grown in the presence of 16 mM NO3 - 1. The early steps of the infection process, including microsymbiont attachment to host root hairs 1,1 z and root hair deformation and infection 7,10,12, are inhibited when seedlings are grown in hydroponic culture containing critical concentrations of nitrate. If the rhizobia infect the root hairs before these levels of nitrate are added, then nodules will form and prematurely senesce 7,12 The biochemical basis for the inhibitory effect of combined nitrogen on the infection of clover root hairs by R. trifolii is just beginning to be understood. Supplying combined nitrogen (NO3 -, NH~ +) to growing roots over a period of 12 h~4 days diminishes both the immunologically detectable levels of trifoliin A, a Rhizobium-binding lectin on clover root hairs, and the selective attachment of R. trifolii 0403 to these root hairs 1. Thus, the presence of combined nitrogen during plant growth plays an important role in regulating these early recognition steps. Hypotheses to explain how nitrate supply regulates levels of trifoliin A on clover root surfaces include i) direct ligand binding of nitrate to trifoliin A resulting in its inactivation, ii) alteration of 'wall receptors' involved in accumulation of trifoliin A on the root surface and iii) inhibition of de novo 259

An inhibitor of murine stem cell proliferation produced by normal human bone marrow

Media conditioned by normal human bone marrow cells contain a specific inhibitor of haemopoietic stem cell proliferation. Molecular ultrafiltration and dose response studies indicate that it is similar to a previously described factor obtained from freshly isolated or long-term cultured murine bone marrow cells. It is suggested that the mechanisms involved in the control of murine and human stem cell proliferation may be essentially identical.

Actions of the haemopoietic stem cell proliferation inhibitor

An inhibitor of haemopoietic stem cell proliferation has been prepared (a) by making extracts of haemopoietic tissue containing slowly proliferating colony-forming units-spleen (CFU-S) and fractioning by molecular ultrafiltration; and (b) by preparing “conditioned” supernatant media of such tissue. Dose-response studies of the effects of these inhibitors on the proportion of proliferating CFU-S which are synthesizing DNA have been carried out for different periods of exposure to the inhibitor. It was found that there is a minimum threshold dose for effective inhibition which varies inversely with the duration of exposure to the inhibitor. Two possible models to explain these results are suggested. First, at the threshold dose, the inhibitor is effective only at the start of the G 1 phase. This permits the G 1 cells present to progress normally into the S phase and, therefore, prevents an immediate observation of the inhibition. As the dose is increased, the effect is extended throughout the G 1 phase to the G 1-S boundary, effectively blocking the entry of cells into the S-phase and resulting in immediate inhibition. Second, the threshold dose is effective early in the G 2 period, which is long compared to the G 1 phase, but the affected cells continue their progress only up to the end of that cycle. The block, therefore, is not seen until this time has elapsed. Higher doses of inhibitor affect later G 2 cells, also resulting in early recognition of the block. The effects of the inhibitor are not lost simply by washing the cells, so that once it has become effective the stem cells remain in a switched off state until such time as they are switched on again by an applied proliferation stimulator.