Sequential Gel Research Articles

Pressure propagation and flow restart in a pipeline filled with a gas pocket separated rheomalaxis elasto-viscoplastic waxy gel

A clogged waxy crude oil pipeline, entails a high pressure to de-structure the gelled oil and resume its normal operation. Our earlier work, which utilized generalized Newtonian fluid based model, demonstrated that the presence of a gas pocket in-between the gel plugs, delays pressure propagation in the downstream gel plug [L. Tikariha, L. Kumar, Pressure propagation and flow restart in the multi-plug gelled pipeline, Journal of Fluid Mechanics. 911 (2021)]. This delay in pressure propagation is effectively utilized for sequential gel degradation. In the present work, we consider gel as a rheomalaxis elasto-viscoplastic (REVP) fluid, capable in predicting flow stoppage and pressure signal stoppage. The governing equations for mass, momentum balance, and phase equation are solved together with the REVP rheology model. The propagation of pressure in a REVP gel-filled pipeline is directly related to the gel material's elastic response. For a REVP gel, the flow-restart in a gelled pipeline depends on the conditional statement γ>γs (i.e. mean gel deformation overcoming the yield strain). In the case of a single gel plug, no-pressure propagation is predicted at intermediate compressibility, whereas pressure propagation without flow restart is predicted at low compressibility. In these cases, the presence of a small gas pocket instigates the gel breakage process, resulting in a flow restart. The gas pocket delays the pressure propagation in the second gel plug, resulting in a pressure gradient higher than yield stress across the first gel plug. The first gel plug, which deforms under a high-pressure gradient, compresses the gas pocket. This results in a larger mean deformation in the first gel plug, and it overcomes the threshold yield strain limit (γs). At strain values larger than yield strain the gel structure breaks, resulting in a lower elastic strength in the first gel plug. The lower elastic gel strength ascertains a lower pressure gradient in the first gel plug, allowing a largely unattenuated pressure to propagate in the second plug. The propagation of unattenuated pressure in the second gel plug results in a pressure gradient higher than the yield stress. This causes sequential gel breakage, resulting in a successful flow restart in a pipeline longer than a critical length. The present study also demonstrates that the pressure propagation and initial transient flow depend on the gel properties (i.e. gel strength and compressibility) and the number and size of gas pockets in the gel. The insights gained from the present work provide a cost-effective solution to mitigate the flow restart problem.

Pressure propagation and flow restart in the multi-plug gelled pipeline

An understanding of pressure propagation mechanisms is crucial in finding suitable conditions for the temporary storage, transmission and attenuation of a large pressure signal in a multi-plug gelled pipeline. The current work investigates the transient flow start in a multi-plug gelled pipeline, filled with a weakly compressible, thixotropic waxy gelled crude oil. A multi-plug gel can form either naturally or artificially. We solve time-dependent mass and momentum conservation equations for the gas phase and conservation equations together with a shear-history-dependent constitutive model for the gel phase to capture the transient phenomena. An advection equation traces the motion of the gel–gas interface using the volume of fluid method. The results of this work show that pressure propagation in the first gel plug compresses it. The compressed first gel plug further compresses the gas pocket, which delays pressure propagation in the downstream gel. The delay in the pressure propagation through the second gel plug creates a high-pressure gradient in the first gel plug. A high-pressure gradient in the first gel results in faster degradation of the first gel. Degraded gel offers a substantially lower resistance to flow, allowing a high-pressure gradient in the second gel. A steeper pressure gradient assists the breakage of the next gel plug and results in a sequential gel breakage. This sequential gel breakage allows flow restart in a long pipeline. The results also indicate that the compressibility of the gas pocket dominates the overall compressibility of the system, controlling both pressure propagation and flow-restart time.

FIRST REPORT OF DAHLIA LATENT VIROID IN TURKEY

Dahlia (Dahlia sp.) is a host of dahlia latent viroid (DLVd), but no information is available on the occurrence of this viroid in Turkey. Thirty asymptomatic dahlia plants were randomly selected in gardens of the cities of Gaziantep and Adana (Turkey) for this study. Total RNA was extracted from approximately 200 mg of leaf tissue of the sampled plants with AxyPrep Multisource Total RNA Midiprep Kit (Axygen Biosciences, USA) and screened by RT-PCR with primers DLVd-P1 (5'-GGGGCTCCTCAGAGAGTCTC-3') and DLVd-P2 (5'- GGGGCAACTCCGAGAGTGCTG-3') (Verhoeven et al., 2013). Twelve plants proved to be infected with DLVd. The amplified DNA bands of the expected size (ca. 340 nucleotides) were purified and directly sequenced. The sequence of five DLVd genetic variants from Turkey showed 98 to 100% maximum nucleotide identity with a genetic variant from the Netherlands (GenBank accession No. JX263426). The occurrence of DLVd in dahlia was confirmed by viroid purification and sequential polyacrylamide gel electrophoresis (Semancik et al., 1988). To our knowledge, this is the first report of DLVd on dahlia plants in Turkey.

Purification and characterization of two novel angiotensin I-converting enzyme inhibitory peptides derived from R-phycoerythrin of red algae (Bangia fusco-purpurea)

R-phycoerythrin prepared from red algae (Bangia fusco-purpurea) was hydrolyzed by pepsin followed by trypsin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides. The IC50 of the hydrolysate of R-phycoerythrin (HRPE) was 191.1 ± 4.1 μg/mL, and the molecular weight of most products (89.9 %) was below 2 kDa. After sequential gel permeation and reversed-phase chromatography steps to purify the hydrolysate, two peptides with the sequences of ALLAGDPSVLEDR and VVGGTGPVDEWGIAGAR were obtained and their IC50 values were 57.2 ± 5.0 and 66.2 ± 4.2 μg/mL, respectively. The ALLAGDPSVLEDR and VVGGTGPVDEWGIAGAR peptides were derived from the β- and α-subunit of R-phycoerythrin from Polysiphonia urceolata, with a 92.3 % (12/13) and 94.1 % (16/17) match, respectively. Both peptides were resistant to digestion by proteinases common in the gastrointestinal tract. Therefore, the identified novel peptides derived from R-phycoerythrin may be used as potential nutraceuticals for development of functional foods.

Selection at the Esterase-2 locus of Drosophila buzzatii? Perturbation-reperturbation experiments.

Apparent selection affecting starch gel electrophoretic alleles at the Esterase-2 locus of Drosophila buzzatii has been detected in laboratory and natural populations. Perturbation-reperturbation of allele frequencies in replicated laboratory populations attempts to test direct selective effects at the locus versus effects of linked loci. Sequential gel electrophoresis has identified more alleles within starch classes, and three of these alleles (within the a, b and c starch alleles) were used in cage population experiments. Allele a/1.00/1.00/1.00 was set up in 10 replicate populations with allele c/1.00/1.00/1.00, and in an independent 10 replicate populations with allele b/0.99/1.01/1.00. For each set, three reperturbations were done. Replicate populations generally showed similar patterns of allele frequency change and clear directionality: effects of selection, not drift. However, four populations deviated from their replicates, indicating dissipation of linkage disequilibrium. Estimates of pre-adult viability in the F2 of pair-wise crosses among 12 sequential gel electrophoretic alleles showed very variable modes of inheritance and relative viability fitnesses. Together with the diversity of patterns of allele frequency change in the cage populations, these results suggest a gene complex, with selection acting on an interacting set of loci which may include Esterase-2.

Alternative Luminal Activation Mechanisms for Paneth Cell α-Defensins

Paneth cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(-/-) mice lack mature α-defensins in Paneth cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(-/-) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(-/-) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(-/-) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(-/-) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.

A Bacteriocin Produced by <i>Lactobacillus brevis</i> KLDS1.0373 Isolated from “Jiaoke”, a Traditional Fermented Cream from China

A bacteriocin produced by Lactobacillus brevis KLDS1.0373 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China was reported in this article. The bacteriocin was partially purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography, and the apparent molecular weight of the partially purified bacteriocin was estimated at approximately 3.8 kDa.

Unique Thermo-Induced Sequential Gel−Sol−Gel Transition of Responsive Multiblock Copolymer-Based Hydrogels

Unique Thermo-Induced Sequential Gel−Sol−Gel Transition of Responsive Multiblock Copolymer-Based Hydrogels

Plantaricin MG active against Gram-negative bacteria produced by Lactobacillus plantarum KLDS1.0391 isolated from “Jiaoke”, a traditional fermented cream from China

A bacteriocin named plantaricin MG produced by Lactobacillus plantarum KLDS1.0391 which was isolated from “Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China is reported in this article. Plantaricin MG was purified by ammonium sulfate precipitation followed by sequential gel filtration chromatography and reverse-phase chromatography. Mass spectrometry analysis showed the mass of plantaricin MG to be approximately 2180 Da. The bacteriocin showed a broad inhibitory activity against Gram-positive and Gram-negative bacteria including Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli. The bacteriocin was extremely heat-stable (30 min at 121 °C) and remained active after incubation at pH 2.0–10.0. It was found to be sensitive to proteolytic enzymes (pepsin, trypsin, papain, α-chymotrypsin, proteinase K, Neutrase and Alcalase). The mode of action of plantaricin MG was identified as bactericidal.

Protein Profiling of Plasma Membranes and Lipid Rafts Defines Aberrant Signaling Pathways in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.

Helicobacter pylori neutrophil-activating protein promotes myeloperoxidase release from human neutrophils

Helicobacter pylori infection induces acute and chronic inflammation and plays a key role in gastric mucosal diseases. H. pylori neutrophil-activating protein (HP-NAP), one of its virulence factors, induces not only chemotactic but also oxidative burst responses of neutrophils. Activated neutrophils use myeloperoxidase (MPO) to generate many cytotoxic oxidants, which might result in gastric mucosal injury. In this study, we evaluated whether HP-NAP could promote MPO release from human neutrophils. Recombinant HP-NAP expressed in Escherichia coli was purified by two sequential gel filtration chromatographies and then subjected to syringe filtration for endotoxin removal. The purified recombinant HP-NAP assembles into a multimer with a secondary structure of the typical α-helix. In addition to stimulating the production of reactive oxygen species, HP-NAP is able to induce the secretion of MPO in human neutrophils. The increased MPO release from neutrophils induced by HP-NAP may be related to the pathogenesis of H. pylori-associated gastritis.

Plasma kallikrein is activated on dermatan sulfate and cleaves factor H

When human plasma is applied to a dermatan sulfate column, amidase activity is detected in the bound fraction and complement factor H is cleaved [A. Saito, H. Munakata, Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate—mediated protease that cleaves factor H, J. Biochem. 137 (2005) 225–233]. Here, the amidase-active fraction was purified by sequential gel filtration and hydroxyapatite chromatography, and the amidase-active protein was identified to be plasma kallikrein by mass spectrometry. The activation of plasma kallikrein was further investigated by Western blotting using plasma deficient in prekallikrein or coagulation factor Xll. The dermatan sulfate column-bound fraction of the prekallikrein- and factor Xll-deficient plasmas did not show any amidase activity and factor H remained intact. Addition of kallikrein, but not activated factor Xll, to factor H purified from plasma resulted in cleavage of factor H. Thus, dermatan sulfate induces contact activation and activates kallikrein-mediated cleavage of FH.

Immunostimulatory effects of oriental plum (Prunus salicina Lindl.)

The fruit of the plum tree (Prunus salicina Lindl.) has been used as a traditional medicinal food in humans to enhance immunity against infectious agents and to treat cancers. However, limited information exists on the mechanisms responsible for its immune enhancing properties. In this study, the immunostimulatory effects of a methanol extract of plum fruit following methanol evaporation and dissolving in PBS were assessed by in vitro lymphocyte proliferation, tumor cell cytotoxicity, and nitric oxide (NO) production. The crude methanol extract stimulated spleen lymphocyte proliferation and NO production by cultured macrophages, and inhibited the viability of tumor cells, significantly greater than media controls. Sequential gel filtration chromatographic separation of the extract on Sephadex G-25 and Sephacryl S-200 gel filtration columns resulted in a more purified preparation that retained the ability to induce lymphoproliferation, tumor killing, and NO production. These results suggest that Prunus salicina contains immunostimulatory components that potentially may be useful in human and veterinary medicine.

Immune Enhancing Properties of Safflower Leaf (Carthamus tinctorius) on Chicken Lymphocytes and Macrophages

Safflower (Carthamus tinctorius) has been used as a traditional medicinal plant to enhance natural immunity and treat cancers. However, limited information exists on the mechanisms responsible for its immune enhancing properties. In this study, the immunostimulatory effects of a methanol extract of safflower leaf were assessed by in vitro lymphocyte proliferation, tumor cell cytotoxicity, and nitric oxide production by cultured chicken macrophages. A crude methanol extract of safflower leaf stimulated spleen lymphocyte proliferation and nitric oxide production, and inhibited the viability of tumor cells significantly greater than medium controls. Sequential gel filtration chromatographic separation of the methanol extract on Sephadex G-25 and Sephacryl S-200 columns resulted in a partially purified preparation that retained the ability to induce lymphoproliferation, tumor killing, and NO production. These results demonstrate for the first time that safflower leaf contains immunostimulatory and anti-tumor component(s) that may be potentially useful as a dietary immunomodulator for poultry.

Preparation of a novel antivenom against Atractaspis and Walterinnesia venoms

The two deadly snakes, Walterinnesia aegyptia (black desert cobra) and Atractaspis microlepidota (mole viper) share a common habitat in the central, eastern and western provinces of Saudi Arabia. Bites by either snake were characterized by rapid death, sometimes before reaching any medical facility. Confusing reports of “a black snake bite” are frequently found. The NAVPC had succeeded in preparing a highly effective antivenom against W. aegyptia venom which is now available in the market, but no antivenom against Atractaspis venom is found worldwide. This is probably because of the low molecular weight of sarafotoxins in the venom and hence their poor antigenic properties. At the NAVPC, sarafotoxins were separated by sequential gel filtration of A. microlepidota venom, while toxin T III of W. aegyptia venom obtained by cation exchange chromatography and gel filtration. Conjugation of the two toxins was carried out using glutaraldehyde in a two-step procedure followed by exhaustive dialysis. The conjugate was utilized to hyperimmunize 3-years old horses for 10 months, applying a low-dosage protocol and immunostimulants; the crude venoms of both snakes being added during the last 2 months. The F( ab′) 2 fraction of the antivenom was obtained by pH-guided salt precipitation, enzyme digestion and tangential desalting and filtration. The bivalent antivenom obtained protected mice and rats against the lethal effects of both venoms and rescued the rats challenged with lethal doses of the venoms in recovery experiments. It also neutralized the haemorrhagic, necrotizing and the cardiotoxic effects of A. microlepidota venom and the neuromuscular blocking effect of W. aegyptia venom. The antivenom offers a good rescue potential to those who are bitten by “a black snake” in Saudi Arabia.

A new method for purification of human plasma retinol-binding protein and transthyretin.

Retinol is transported in the blood bound to a specific carrier protein called retinol-binding protein (RBP), which in turn binds to another protein, transthyretin (TTR), a homotetrameric, thyroid-hormone-transporting protein. Binding of TTR increases the apparent molecular mass of RBP and thereby prevents glomerular filtration of RBP. Owing to their rapid turnover rates, plasma concentrations of these proteins are sensitive indicators and valuable diagnostic markers of vitamin A nutrition, protein energy malnutrition, infection and renal-tubule function. Previously RBP and TTR were purified by using different procedures, either from plasma or from pathological urine of humans. In general the procedure for purification of RBP and TTR is laborious, and extensive sample recycling is necessary for purification in appreciable amounts. In the present study, we have purified RBP using a simple method, which involves (NH(4))(2)SO(4) fractionation followed by sequential gel filtration under native conditions and 6 M urea. TTR, which was eluted in 60 kDa fractions during urea/Sephadex G-100 chromatography, was further purified to homogeneity using a combination of two dye-affinity chromatographic steps on Reactive Yellow and Cibacron Blue coupled to agarose columns. SDS/PAGE and immunoblotting, apart from typical UV absorption and fluorescent properties of RBP, were used for characterizing the purified RBP and TTR. Furthermore, the purified RBP and TTR were found to be functional from mutual binding monitored by fluorescence quenching.

Analysis of Steady-state Protein Phosphorylation in Mitochondria Using a Novel Fluorescent Phosphosensor Dye

The phosphorylation of mitochondrial proteins is pivotal to the regulation of respiratory activity in the cell and to signaling pathways leading to apoptosis, as well as for other vital mitochondrial processes. A number of protein kinases have been identified in mitochondria but the physiological substrates for many of these remain unknown or poorly understood. By necessity, most studies of mitochondrial phosphoproteins to date have been conducted using in vitro incorporation of 32P. However, proteins that are highly phosphorylated from in situ reactions are not necessarily detected by this approach. In this study, a new small molecule fluorophore has been employed to characterize steady-state levels of mitochondrial phosphoproteins. The dye is capable of sensitive detection of phosphorylated amino acid residues in proteins separated by gel electrophoresis. When the fluorescent dye is combined with a total protein stain in a sequential gel staining procedure, the phosphorylated proteins can be visualized in the same gel as the total proteins. To optimize resolution of the proteins in mitochondria, a previously described sucrose gradient fractionation method was employed prior to gel electrophoresis. Phosphorylated proteins, as defined by the fluorescence of the phosphosensor, were excised from the gels and identified by peptide mass fingerprinting. One novel and prominent phosphoprotein identified in this manner was determined to be the 42-kDa subunit of mitochondrial complex I.

Two novel myogenic factors identified and isolated by sequential isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A number of environmental factors were used experimentally to enhance myogenesis during muscle regeneration; however, many hormones and growth factors have been shown to have the ability to increase the rate of satellite cell division, but they only work on satellite cells that are already active in many animal experiments. Recently, the crushed muscle extract (CME) of rats was found to be able to trigger dormant adult rat satellite cells to re-enter the cell mytogenic cycle; however, the identity of the active factors present in rat CME remains unknown. In the present study, the CME was fractionated by the strategy of sequential isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with functional analysis by myoblast culture. Two satellite cell-specific myogenic factors were identified and purified from CME by this strategy. One of the factors has a molecular mass of around 7 kDa and another about 39 kDa. The factor of 39 kDa could be retained in heparin-Sepharose column and eluted with phosphate-buffered saline (PBS) containing 1 M NaCl, but the 7 kDa factor did not bind to the heparin column. These two purified myogenic factors could synergistically trigger the proliferation and differentiation of dormant satellite cells, whose progenies subsequently fuse in vitro, or fuse to pre-existing partially damaged muscle fibers to form full repair of the damaged muscle fibers or to form new myotubes to replace the completely damaged muscle fibers during the cascade of muscle healing and regeneration in vivo. The identities of these two myogenic factors are under study.

Partial Preparative Purification of Leptine I from Foliage of the Wild Potato, Solanum Chacoense (BITT.)

ABSTRACT Leptine I, a glycoalkaloid only known to occur in the foliage of the wild potato species Solanum chacoense (Bitt.), is a potent feeding deterrent to the economically serious insect pest, the Colorado potato beetle (Leptinotarsa decemlineata Say). In order to demonstrate, systematically, the effectiveness of leptine I, incorporation into synthetic beetle diet trials is necessary. We describe a preparative procedure for the partial purification of leptine I by a series of steps, starting with a solid-phase C18 extraction, followed by sequential silica gel chromatography, and finally reversed-phase preparative HPLC. This preparation yields a white powder, containing leptine I as the sole glycoalkaloid, with an overall purity of greater than 65%, and is entirely suitable for incorporation into synthetic diets.

Isolation of Human Salivary Mucin MG2 by a Novel Method and Characterization of Its Interactions with Oral Bacteria

Human salivary mucin MG2 was purified from submandibular/sublingual gland secretion by ultrafiltration and sequential gel filtration chromatography on Sephadex G-200, Superose 6 (prepgrade), and Superose 6. This method differs from earlier procedures in that all steps are performed in the presence of 4 M guanidine hydrochloride and do not involve covalent modification of the mucin molecule. Electrophoretic analyses and Western blotting showed that purified MG2 did not contain detectable levels of other salivary proteins. Amino acid analysis showed that the composition of purified MG2 was in excellent agreement with the deduced sequence of MG2 apomucin encoded in the MUC7 gene. The yield of purified MG2 was 10–15 mg from 750 ml of salivary secretion. Binding of purified MG2 toStreptococcus mutans in vitrowas not significantly affected by reductive methylation, but was nearly abolished by reduction and alkylation. These data identified a functional determinant for mucin–bacterial interactions in the N-terminal region where the only two cysteines (Cys45and Cys50) in the MG2 apomucin occur. Additionally, purified MG2 bound to four strains of oralStreptococci,indicating that the binding is not dependent on complexing with other salivary proteins, such as secretory immunoglobulin A. The purification procedure described in this work will facilitate investigation of the role of MG2 in the oral environment.