High-resolution Gel Filtration Research Articles

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Abstract 445: Phospholipid Levels Associated with Specific High-Density Lipoprotein Subfractions Predict Cardiovascular Risk in Young Adults with Type 2 Diabetes

Risk for atherosclerosis is greatly increased in people who have type 2 diabetes (T2D). Because of this, the emerging epidemic of adolescent T2D holds ominous implications for premature cardiovascular disease (CVD). High density lipoprotein cholesterol (HDL-C) represents the body’s natural defense against CVD but its levels are depressed in individuals with T2D. Recent studies indicate HDL exists as distinct subspecies raising the possibility that certain species may be more cardioprotective than others. However, little is known regarding the role of HDL subspecies in T2D, especially in the adolescent population. Thus we sought to evaluate HDL subspecies and determine whether certain subspecies are associated with protection against the development of early atherosclerosis as measured by carotid intima medial thickness (IMT). Healthy controls and youth with T2D were recruited. Whole plasma was analyzed by high-resolution gel filtration chromatography to resolve HDL sized particles and lipids in each fraction were quantitated by colorimetric assay. T-tests were used to evaluate group differences and linear regression models were constructed to determine independent predictors of carotid IMT. Youth with T2D had higher BMI, total cholesterol and lower HDL-C compared to healthy controls, p<0.05. The groups did not differ in LDL-C, triglycerides or BP. Phospholipid distributions of HDL subspecies were found to be shifted in participants with T2D compared to controls (p<0.05). There was a significant inverse correlation between carotid IMT and the phospholipid content of larger HDL subfractions 22-24 (p<0.05) in youth with T2D. Linear models demonstrate HDL fraction 22 was the only independent predictor of carotid IMT while HDL-C, LDL-C, total -C and triglycerides were not significant. These data suggest an altered HDL particle subclass distribution may better predict protection against early atherosclerosis. Thus analyzing the HDL subspecies may be a more powerful approach to assessing cardiovascular risk than the currently accepted standard of HDL-C.

Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-β-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 μM ml(-1) and 0.119 μM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.

Partial purification of an antifungal protein produced byEnterococcus faecalis CHD 28.3

The antifungal protein produced byEnterococcus faecalis CHD 28.3 isolated from the Cheddar cheese was partially purified from the culture supernatant using ultrafiltration, anion exchange and gel filtration chromatography. Employing a 10 kDa cut off membrane, ultrafiltration of the culture supernatant resulted in a recovery of 44.6% of the antifungal protein with 1.7 fold increase in the specific activity. Anion-exchange chromatography using DEAE-Sepharose matrix followed by purification of the samples using high resolution gel filtration chromatography employing Superose-12 column/FPLC system led to a very low (∼0.5%) recovery of antifungal activity with an increase in specific activity by 11.9 fold relative to initial value in crude supernatant. The molecular mass of the antifungal protein from the high resolution gel filtration was estimated to be around 11 KDa. The partially purified protein obtained after DEAE-Sepharose chromatography step was completely inactivated by heat treatment at 72 °C for 15 seconds.

Small molecular weight proteins/peptides present in the in vivo formed human acquired enamel pellicle

Objective The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle. Design Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides <10 kDa obtained by both methods were analyzed by LC–ESI-MS/MS. Results 78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins. Conclusions The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.

Protection of IgE‐mediated allergic sensitization by active immunization with antigenized IgE B‐cell epitope in GFP protein scaffold

Green fluorescent protein (GFP) exhibits a rigid central β‐barrel, formed by eleven β‐strands with floppy loops spanning between the stands. The spanning loop, the site 6 of GFP, was engineered with RE cloning sites for inserting oligonucleotides corresponding to FcεRI‐binding sequence of human IgE. In a high throughput format, oligonucleotides encoding eight amino acids of the receptor‐binding regions were inserted in an expression cassette was assembled with 5’ in vitro transcription and translation enabling sequences. Antigenized C2‐3 linker (C2‐3L) was shown to react strongly by immunoblotting with polyclonal anti‐IgE under native gel electrophoresis and transfer. Recombinant C2‐3L antigenized GFP, expressed in recombinant vector was purified to homogeneity by metal affinity column, followed by Sephacryl S‐200 high resolution gel filtration. Hyperimmune sera from mice immunized with C2‐3L antigenized GFP exhibit high titers of anti‐IgE reactive with JW8 murine/human chimeric IgE. Further, elevated serum anti‐C2‐3L and affinity pure antibodies effectively inhibits binding of JW8 IgE to recombinant FcεRIα. and desensitizes JW8 to rat RBL‐2H3 transfected with human FcεRIα. This observation raises the distinct possibility that active IgE vaccine may indeed be employed in raising active protective anti‐IgE in allergic patients as an alternative to passive immunization with MAb‐E25 anti‐IgE. Moreover, this antigenized epitope (s) platform may be extended to analyzing specific B‐cell epitopes of other proteins of interest.

Plasma Adiponectin Complexes Have Distinct Biochemical Characteristics

Adipocytes release the secretory protein adiponectin in a number of different higher-order complexes. Once synthesized and assembled in the secretory pathway of the adipocyte, these complexes circulate as biochemically distinct and stable entities with little evidence of interchange between the different forms that include a high-molecular-weight (HMW) species, a hexamer (low-molecular-weight form), and a trimeric form of the complexes. Here, we validate a high-resolution gel filtration method that reproducibly separates the three complexes in recombinant adiponectin and adiponectin from human and murine samples. We demonstrate that the HMW form is prominently reduced in male vs. female subjects and in obese, insulin-resistant vs. lean, insulin-sensitive individuals. A direct comparison of human and mouse adiponectin demonstrates that the trimer is generally more abundant in human serum. Furthermore, when the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation. The complex distribution of adiponectin can be regulated in several ways. Both mouse and human HMW adiponectin are very stable under basic conditions but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However, the lack of calcium binding results in a destabilization of the structure. Disulfide bond formation (at position C39) is also important for complex formation. A mutant form of adiponectin lacking C39 prominently forms HMW and trimer but not the low-molecular-weight form. Injection of adiponectin with a fluorescent label reveals that over time, the various complexes do not interconvert in vivo. The stability of adiponectin complexes highlights that the production and secretion of these forms from fat cells has a major influence on the circulating levels of each complex.

Protection of IgE-mediated allergic sensitization by active immunization with IgE loops constrained in GFP protein scaffold

Green fluorescent protein (GFP) exhibits a rigid central β-barrel, formed by eleven β-strands with floppy loops spanning between the stands. Herein, we evaluate whether the rigid β-barrel may serve as a scaffold that can constrain the loops of a foreign protein, and thus its antigenicity. The spanning loops, site 6 of GFP, were engineered with RE cloning sites for inserting oligonucleotides corresponding to FcɛRI-binding sequence of human IgE. In a high-throughput format, shortened oligonucleotides encoding eight amino acid residues of the receptor-binding regions were inserted into site 6 of GFP by PCR, followed by enabling sequences for in vitro transcription and translation at the 5′ end. Antigenized C2-3 linker (C2-3L) was shown by immuno-blots with polyclonal anti-IgE under native gel electrophoresis and transfer. Recombinant antigenized GFP was expressed and purified to homogeneity by metal affinity column, followed by Sephacryl S-200 high resolution gel filtration. Hyperimmune sera from mice immunized with C2-3L antigenized GFP contain anti-IgE reactive with JW8 murine/human chimeric IgE. Further, elevated serum anti-C2-3L and affinity pure antibodies effectively inhibits binding of JW8 IgE to recombinant FcɛRIα, and desensitizes JW8 to rat RBL-2H3 transfected with human FcɛRIα. This observation raised the possibility that active IgE vaccine may be employed in raising active protective anti-IgE in allergic patients as an alternative to passive immunization with MAb-E25 anti-IgE. Taken together, GFP appears suitable protein scaffold for spanning/constraining the C2-3L of human IgE as active vaccine; and this technique may be generally employed for eliciting antibodies to specific B-cell epitopes of other proteins.

Cellular Adhesion Responses to the Heparin-binding (HepII) Domain of Fibronectin Require Heparan Sulfate with Specific Properties

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.

Native tubulin-folding cofactor E purified from baculovirus-infected Sf9 cells dissociates tubulin dimers

Tubulin-folding cofactor E (TBCE) is an α-tubulin-binding protein involved in the formation of the tubulin dimer and in microtubule dynamics, through the regulation of tubulin heterodimer dissociation. TBCE has also been implicated in two important related human disorders, the Kenny–Caffey and Sanjad–Sakati syndromes. The expression of TBCE as a recombinant protein in bacteria results in the formation of insoluble inclusion bodies in the absence of denaturing agents. Although the active protein can be obtained from mammalian tissues, biochemical studies of TBCE present a special challenge. To express and purify native TBCE, a recombinant baculovirus expression system was used. Native wild-type TBCE purified from Sf9 extracts was sequentially purified chromatographically through cation exchange, hydrophobic interaction, and high-resolution gel-filtration columns. Mass spectrometric analysis identified 30% of the sequence of human TBCE. A stoichiometric excess of purified TBCE dissociated tubulin heterodimers. This reaction produced a highly unstable TBCE–α-tubulin complex, which formed aggregates. To distinguish between the aggregation of tubulin dimers induced by TBCE and tubulin dissociation, TBCE and tubulin were incubated with tubulin-folding cofactor A (TBCA). This cofactor captures the β-tubulin released from the heterodimer with a stoichiometry of 1:1, as previously demonstrated. The β-tubulin polypeptide was recovered as TBCA–β-tubulin complexes, as demonstrated by non-denaturing gel electrophoresis and specific antibodies directed against β-tubulin and TBCA.

Purification of the Small Mechanosensitive Channel of Escherichia coli (MscS): the Subunit Structure, Conduction, and Gating Characteristicsin Liposomes

The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of ∼200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20–60mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (γ). Po(γ) curves had the midpoint at 5.5±0.1 dyne/cm and gave estimates for the energy of opening ΔG=11.4±0.5 kT, and the transition-related area change ΔA=8.4±0.4nm2 when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

Adherence of vancomycin to proteins.

Vancomycin possesses the unusual property of promoting the aggregation of proteins. It also binds to itself (dimerization). Both properties may be related to its antimicrobial activity and we report here procedures to measure them. The position of the negative ellipticity band in the near ultraviolet circular dichroism spectrum of the vancomycin monomer shifts as a function of antibiotic concentration and can be used to readily determine the monomer-dimer equilibrium constant. These measurements complement those performed by high-resolution gel filtration to measure the same process. Aggregation of purified proteins was determined by turbidity measurements. Both dimerization and protein aggregation are influenced by anions whose effectiveness is related to their carboxyl pKa values, thus linking these two properties.

Evidence for production of a new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79: characterization of the structural gene encoding butyrivibriocin OR79A.

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5' region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.

Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 °C in the presence of Na+ ions but the structure is stabilized up to 75 °C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 °C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd

The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

Processing of Preproricin in Transgenic Tobacco

The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS–PAGE gel of lactose–agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.

Noxious Components of Venom from the Pupa-Specific ParasitoidPimpla hypochondriaca

Venom from the endoparasitoid waspPimpla hypochondriacainduced a lethal paralysis when injected into the hemocoel of larval, pupal, and adult stages of the tomato mothLacanobia oleraceaand was also toxic to the adult stages of the houseflyMusca domesticaand the cockroachBlatella germanica.Heating the venom to 58°C did not reduce its potency againstM. domestica.Phenoloxidase activity was detected in diluted venom using dihydroxyphenylalanine as the substrate and occurred independently of activators. Enzyme activity was strongly inhibited by 2.0 μM phenylthiocarbamide (PTC), a known inhibitor of phenoloxidase, and was not detectable in venom that had been heated to 58°C for 10 min. Venom treated to remove phenoloxidase was shown to inhibit phenoloxidase activity in untreated venom, with a potency likely to be sufficient to prevent enzyme activity in the parasitoid's venom sac. The venom was found to be cytotoxic to an insect cell line (SF21) fromSpodoptera frugiperda,and this activity was not reduced when the cells were cultured in the presence of 20 μM PTC. Venom constituents were separated according to size using high-resolution gel filtration. Three venom components: phenoloxidase, a factor which caused paralysis ofM. domestica,and a factor which was cytotoxic to SF 21 cells were detected and found to have maximum activity in fractions 19, 40–42, and 50–51, respectively. Phenoloxidase and the factor causing paralysis ofM. domesticahad estimated apparent molecular masses of 390 and 27 kDa, respectively, while the cytotoxic factor had an apparent molecular mass of less than 13 kDa. Assay for inhibitors of venom phenoloxidase identified two major inhibitory components having highest activity in fractions 48 and 60, respectively. The larger of these two factors had an estimated apparent molecular mass of 12 kDa, but the other factor was considerably smaller than the lowest molecular mass standard (13.7 kDa) used to calibrate the column.

Chromatographic removal and heat inactivation of hepatitis B virus during the manufacture of human albumin.

The purpose of the present study was to examine the efficacy of the chromatographic and pasteurization steps, employed in the manufacture of human albumin, in the removal and/or inactivation of hepatitis B virus (HBV). Most human albumins manufactured today are prepared from donor plasma by fractionation methods that use precipitation with cold ethanol. CSL Limited, an Australian biopharmaceutical company, has recently converted its method of manufacture for albumin from a traditional Cohn fractionation method to a method employing chromatographic techniques. A step-by-step validation of virus removal and inactivation was performed on this manufacturing process, which includes a DEAE-Sepharose(R) and CM-Sepharose(R) Fast Flow ion-exchange step, a Sephacryl(R) S200 High-Resolution gel-filtration step and a bulk pasteurization step where product is held at 60 degreesC for 10 h. HBV partitioning experiments were conducted on scaled-down chromatographic columns with hepatitis B surface antigen (HBsAg) as a marker, whereas the HBV model virus, duck HBV, was used to study the inactivation kinetics during pasteurization. Reductions for HBsAg through the three chromatographic steps resulted in a total log10 decrease of 1.5 log10, whereas more than 6.5 log10 decrease in duck HBV in Albumex(R)5 was achieved during pasteurization.

Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin.

While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-crystallin contains N- and C-terminal arms, associates to form homodimers. In order to answer the question of whether the extensions are involved in dimerisation of chick lens beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension. High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability. Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.