Native High Molecular Weight Research Articles

Digestion and gut-microbiota fermentation of cocoa melanoidins: An in vitro study

This research aimed to compare the in vitro digestibility and fermentability of melanoidins extracted from cocoa nibs fast-roasted with a fluidized bed roaster (FR-melanoidins), melanoidins from cocoa nibs slow-roasted in a convective oven (SR-melanoidins), and native high molecular weight compounds extracted from unroasted cocoa nibs (UR-HMWC). Data showed that both types of melanoidin extracts, especially FR-melanoidins, better release polyphenols during digestion and earlier produce short-chain fatty acids during in vitro fermentation than UR-HMWC. The delayed catabolism of UR-HMWC resembled that of dense fibers, while FR- and SR-melanoidins resembled oligosaccharides. The production of SCFA was positively correlated with the increase in Bacteroides sp. The profile of the genera identified in the proximal colon showed the differences in fermentability between FR- and SR-melanoidins, with FR-melanoidins having the higher relative growth of nonpathogenic bacteria. This study demonstrated the in vitro digestibility of bound phenolic compounds and the potential prebiotic activity of cocoa melanoidins.

Enzymatic depolymerization of the GY785 exopolysaccharide produced by the deep-sea hydrothermal bacterium Alteromonas infernus: Structural study and enzyme activity assessment

Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.

Hyaluronan Isolation from Mouse Mammary Gland.

The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis ( Calabro et al., 2000 ).

Enzymatic and acidic degradation of high molecular weight dextran into low molecular weight and its characterizations using novel Diffusion-ordered NMR spectroscopy

Low molecular weight fractions were derived from native high molecular weight dextran produced by Leuconostoc mesenteroides KIBGE-IB26. Structural characterization of native and low molecular weight fractions obtained after acidic and enzymatic hydrolysis was done using FTIR and NMR spectroscopy. The molecular weight was estimated using Diffusion Ordered NMR spectroscopy. Native dextran (892kDa) is composed of α-(1→6) glycosidic linkage along with α-(1→3) branching. Major proportion of 528kDa dextran was obtained after prolong enzymatic hydrolysis however, an effective acidic treatment at pH-1.4 up to 02 and 04h of exposure resulted in the formation of 77kDa and 57kDa, respectively. The increment in pH from 1.4 to 1.8 lowered the hydrolysis efficiency and resulted in the formation of 270kDa dextran fraction. The results suggest that derived low molecular weight water soluble fractions can be utilized as a drug delivery carrier along with multiple application relating pharmaceutical industries.

Multiwalled Carbon Nanotube Functionalization with High Molecular Weight Hyaluronan Significantly Reduces Pulmonary Injury.

Commercialization of multiwalled carbon nanotubes (MWCNT)-based applications has been hampered by concerns regarding their lung toxicity potential. Hyaluronic acid (HA) is a ubiquitously found polysaccharide, which is anti-inflammatory in its native high molecular weight form. HA-functionalized smart MWCNTs have shown promise as tumor-targeting drug delivery agents and can enhance bone repair and regeneration. However, it is unclear whether HA functionalization could reduce the pulmonary toxicity potential of MWCNTs. Using in vivo and in vitro approaches, we investigated the effectiveness of MWCNT functionalization with HA in increasing nanotube biocompatibility and reducing lung inflammatory and fibrotic effects. We utilized three-dimensional cultures of differentiated primary human bronchial epithelia to translate findings from rodent assays to humans. We found that HA functionalization increased stability and dispersion of MWCNTs and reduced postexposure lung inflammation, fibrosis, and mucus cell metaplasia compared with nonfunctionalized MWCNTs. Cocultures of fully differentiated bronchial epithelial cells (cultivated at air-liquid interface) and human lung fibroblasts (submerged) displayed significant reduction in injury, oxidative stress, as well as pro-inflammatory gene and protein expression after exposure to HA-functionalized MWCNTs compared with MWCNTs alone. In contrast, neither type of nanotubes stimulated cytokine production in primary human alveolar macrophages. In aggregate, our results demonstrate the effectiveness of HA functionalization as a safer design approach to eliminate MWCNT-induced lung injury and suggest that HA functionalization works by reducing MWCNT-induced epithelial injury.

Hyaluronan, Inflammation, and Breast Cancer Progression.

Breast cancer-induced inflammation in the tumor reactive stroma supports invasion and malignant progression and is contributed to by a variety of host cells including macrophages and fibroblasts. Inflammation appears to be initiated by tumor cells and surrounding host fibroblasts that secrete pro-inflammatory cytokines and chemokines and remodel the extracellular matrix (ECM) to create a pro-inflammatory “cancerized” or tumor reactive microenvironment that supports tumor expansion and invasion. The tissue polysaccharide hyaluronan (HA) is an example of an ECM component within the cancerized microenvironment that promotes breast cancer progression. Like many ECM molecules, the function of native high-molecular weight HA is altered by fragmentation, which is promoted by oxygen/nitrogen free radicals and release of hyaluronidases within the tumor microenvironment. HA fragments are pro-inflammatory and activate signaling pathways that promote survival, migration, and invasion within both tumor and host cells through binding to HA receptors such as CD44 and RHAMM/HMMR. In breast cancer, elevated HA in the peri-tumor stroma and increased HA receptor expression are prognostic for poor outcome and are associated with disease recurrence. This review addresses the critical issues regarding tumor-induced inflammation and its role in breast cancer progression focusing specifically on the changes in HA metabolism within tumor reactive stroma as a key factor in malignant progression.

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.

Synthesis and antiherpetic activity of carboxymethylated and sulfated hyaluronan derivatives

Native high molecular weight hyaluronan (HMW-HA, MW=1×106g/mol) and a thermally degraded low molecular weight hyaluronan (LMW-HA, MW=1.3–1.4×105g/mol) were carboxymethylated providing products with degrees of carboxymethylation (DSCM) of up to 0.8. Sulfation of resulting carboxymethyl hyaluronan (CM-HA) and hyaluronan (HA) was performed by different sulfation procedures enabling the control of the degree of sulfation (DSS) in the obtained new carboxymethyl hyaluronan sulfates (CM-HA-S) and hyaluronan sulfates (HA-S), respectively, in a range between 0.9 and 3.3. Both carboxymethylation and sulfation were found to take place preferentially at the primary hydroxyl groups of HA. The antiviral activity of these synthesized HA derivatives was tested against Herpes simplex virus type 1. Both HA-S and CM-HA-S derivatives with high DSS values of about 3.0 exhibit a strong antiherpetic activity. The CM-HA derivatives were found to be not active and an additional effect of introduced carboxymethyl groups on the antiherpetic activity of CM-HA-S derivatives was not observed. In the case of HA-S, the antiviral efficacy can be correlated with the DSS and becomes stronger with increasing DSS values.

High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury

A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

Purification and characterization of a native zinc-binding high molecular weight multiprotein complex from human seminal plasma

The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.

Force spectroscopy of an elastic peptide: Effect of D2O and temperature on persistence length

This study explores the mechanical unfolding of elastic protein analogues as a function of temperature, in both H₂O and D₂O, using atomic force microscopy (AFM) force spectroscopy in a specially constructed AFM liquid cell. This represents the first time that the effect of D₂O on protein flexibility has been investigated at the single molecule level by this technique. Model elastic peptides, R6, were encoded from synthetic genes expressed in Escherichia coli. The peptides possess short N- and C-terminal domains with central repetitive domains containing 13 repeats of the motif PGQGQQ-plus-GYYPTSLQQ. These sequences mimic those in native high molecular weight subunit glutenin proteins which confer elasticity to bread dough. Fitting single molecule stretching events to the worm-like chain model, allows determination of the molecular persistence length under various experimental conditions. The effect of increasing the temperature is to increase the persistence length of the peptide in both H₂O and D₂O, consistent with the expected "thermal softening" effect. However, the effect is significantly enhanced in D₂O, in which the persistence length at 45°C is ∼25% greater than the value measured in H₂O at the same temperature. Stronger intrapeptide H-bonding due to isotopic substitution of hydrogen for deuterium is the most likely cause of the enhanced backbone rigidity.

Acide hyaluronique : structure, métabolisme et implication dans la cicatrisation

Hyaluronan (HA) is a key component of the extra-cellular matrix and is involved in several mechanisms of the wound healing process. It is highly hygroscopic and is involved in the visco-elasticity of the skin. The properties of this molecule depend on its molecular weight. Native high molecular weight HA has structural properties whereas HA degradation products (oligomers) stimulate endothelial cell proliferation and migration. HA oligomers modulate inflammatory processes and promote neo-angiogenesis during the different steps of wound healing. HA mediates its biological effects through binding interactions with specific cell-associated receptors (CD44 and RHAMM). The tridimensional structure of HA is variable and acts as a framework for cell adhesion and migration. Thanks to its different physico-chemical properties, hylaluronic acid is very useful in the biomedical field.

Reversal effects of hyaluronan oligosaccharides on adriamycin resistance of K562/A02 cells

As the interaction of hyaluronan (HA) with its receptor CD44 contributes to multidrug resistance (MDR) of tumor cells, HA oligosaccharides (o-HAs), as HA antagonists, may be useful to reverse the MDR. The objective of this study was to investigate the reversal effects of four o-HAs, including 4 saccharide residue (o-HA4), 6 saccharide residue (o-HA6), 8 saccharide residue (o-HA8), and 10 saccharide residue (o-HA10) fragments, on adriamycin (ADR)-resistant K562/A02 cells. The four o-HAs were prepared by digesting the native high molecular weight HA with hyaluronidase and gel filtration chromatography. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to assess the cytotoxicity of the four o-HAs and/or ADR on K562/A02 and K562 cells. The intracellular accumulation of ADR in K562/A02 cells was measured by flow cytometry. By comparing the IC50 (concentration resulting in 50% inhibition of cell growth) of ADR with K562/A02 cells in the presence and absence of a series of different concentrations of o-HAs, the reversal folds of the four o-HAs were calculated. The reversal folds of o-HA4, o-HA6, o-HA8, and o-HA10 were 2.04, 2.05, 1.91, and 1.84, respectively. After o-HA4, o-HA6, o-HA8, and o-HA10 treatment, the intracellular amounts of ADR were increased to 3.90, 3.92, 3.76, and 3.39 times, respectively. Shorter o-HAs (o-HA4 and o-HA6) showed stronger reversal effects than longer o-HAs (o-HA8 and o-HA10). In conclusion, the results showed that the four o-HAs could effectively reverse the ADR resistance of K562/A02 cells by increasing the intracellular accumulation of ADR. O-HAs may be used as MDR reversal drugs to increase the effectiveness of chemotherapy.

Evaluation of angiogenic activities of hyaluronan oligosaccharides of defined minimum size

Aims Oligosaccharides of hyaluronan (o-HAs) were proved to have pro-angiogenic activities. The aim of this study was to obtain four hyaluronan oligosaccharides (o-HAs) of defined molecular weight including 4 saccharide residues (o-HA4), 6 saccharide residues (o-HA6), 8 saccharide residues (o-HA8), and 10 saccharide residues (o-HA10) under optimum conditions, and compare their angiogenic activities. Main methods The four o-HAs were prepared by digesting the native high molecular weight HA with hyaluronidase under optimum conditions. The effects of the four o-HAs on human umbilical vein endothelial cell (HUVEC) proliferation were measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenil tetrazolium bromide (MTT) assay. Angiogenic effects of the four o-HAs were evaluated using chicken chorioallantoic membrane (CAM) assay. The effects of the four o-HAs on the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) messenger ribonucleic acid (mRNA) expressions were detected by semi-quantitative polymerase chain reaction (PCR). Key findings O-HA6, o-HA8 and o-HA10 could effectively promote the proliferation of HUVEC, induce angiogenesis in the CAM assay and increase VEGF mRNA levels in HUVEC. No significant effects were found with o-HA4. Significance These results suggested that o-HA6, o-HA8, and o-HA10, but not o-HA4 were effective angiogenic factors.

Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing

To determine if oligosaccharides of hyaluronan (o-HA) promotes wound recovery by accelerating angiogenesis and to study the mechanisms by which o-HA stimulates endothelial cell (EC) proliferation. Using hyaluronidase digestion, we prepared a mixture of hyaluronan (HA) fragments sizesd 2 to 10 disaccharides units, and studied their effects on EC growth and migration in mimicking wound recovery in vitro. The effects of o-HA on EC growth in vitro were studied by counting cell numbers. The roles of 2 hyaluronan receptors on EC cells, CD44 and RHAMM (Receptor for HA-Mediated Motility), were studied in initiating signaling cascades, using immunoblot assay. Signal transduction was determined by blocking antibodies to CD44 and RHAMM. An in vitro wound healing model was prepared by scratching the cellular layer of cultured EC, and movement of cells into the denuded area was quantified. o-HA was a strong stimulator to EC proliferation at low concentration 10microg/ml compared with native high molecular weight HA (n-HA) (P < 0.01). Signal transduction may be initiated by o-HA via RHAMM receptor on EC membrane, but not CD44. In the in vitro model, the lesion area was nearly completely recovered when the EC layer was exposed to o-HA 40hrs post-injury, whereas the wound area remained half recovered pretreated with native undegraded large HA and control medium.(P < 0.05 from 24 to 40hrs). Hyaluronan oligosaccharides may play a role in wound healing by increasing angiogenesis. o-HA-RHAMM binding dependent signal transduction pathway may be important in the regulation of angiogenesis associated with EC proliferation.

Preparation and characterization of hyaluronan oligosaccharides for angiogenesis study

Low molecular weight hyaluronan (HA) is proved to be active in stimulating endothelial cell (EC) proliferation. The study is to obtain biological active HA fragments of defined length from high molecular weight HA for angiogenesis research. A novel method, by a high-voltage polyacrylamide-gel electrophoresis (PAGE) that uses specific fluorophore labeling, was developed to identify the degraded products digested from native high molecular weight HA. The polymer HA was digested by hyaluornidase partially, and the enzymatic fragments of HA or HA oligosaccharides (o-HAs) were analyzed for molecular sizes by a combination of PAGE, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The purified HA oligomers were pooled and the biological effects of the mixtures on EC proliferation were studied, by separating the digests of HA fragments by size exclusion chromatography on a Bio-Gel P-10 column. The prepared o-HAs were collected and analyzed by a series of methods, PAGE, HPLC, and MS, which indicated a mixture of oligosaccharides covering a range of different sizes from 4 to 20-mer. The minimal molecular size determined by the special PAGE was 2 disaccharide, and the method can determine o-HA mass as high as 10 disaccharide (seeing the least clear band as much as possible on gel). Fractions containing HA fragments with sizes from 4 to 20-mer were pooled by distinct oligosaccharide species and added to the culture of pig endothelial cell (PIEC) for biological activities study. A significant increase in EC proliferation was found in comparison with nondigested high molecular weight HA. Our results suggested that o-HA can be obtained by a method of fluorophore-labeled olyacrylamide gel electrophoresis for identifying as small as 4-mer oligomer HA from high molecular weight HA-digested products. This method was proved to be sensitive, simple, and rapid in characterizing saccharides, which may be used by replacing the complicated ways of HPLC and MS. The prepared o-HA was found to be active in stimulating EC proliferation.

CD44 stimulation by fragmented hyaluronic acid induces upregulation of urokinase-type plasminogen activator and its receptor and subsequently facilitates invasion of human chondrosarcoma cells.

It has been established that fragmented hyaluronic acid (HA), but not native high molecular weight HA, can induce angiogenesis, cell proliferation and migration. We have studied the outside-in signal transduction pathways responsible for fragmented HA-mediated cancer cell invasion. In our study, we have studied the effects of CD44 stimulation by ligation with HA upon the expression of matrix metalloproteinases (MMPs)-2 and -9 as well as urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) and the subsequent induction of invasion of human chondrosarcoma cell line HCS-2/8. Our study indicates that (i) CD44 stimulation by fragmented HA upregulates expression of uPA and uPAR mRNA and protein but does not affect MMPs secretion or PAI-1 mRNA expression; (ii) the effects of HA fragments are critically HA size dependent: high molecular weight HA is inactive, but lower molecular weight fragmented HA (Mr 3.5 kDa) is active; (iii) cells can bind avidly Mr 3.5 kDa fragmented HA through a CD44 molecule, whereas cells do not effectively bind higher Mr HA; (iv) a fragmented HA induces phosphorylation of MAP kinase proteins (MEK1/2, ERK1/2 and c-Jun) within 30 min; (v) CD44 is critical for the response (activation of MAP kinase and upregulation of uPA and uPAR expression); and (vi) cell invasion induced by CD44 stimulation with a fragmented HA is inhibited by anti-CD44 mAb, MAP kinase inhibitors, neutralizing anti-uPAR pAb, anti-catalytic anti-uPA mAb or amiloride. Therefore, our study represents the first report that CD44 stimulation induced by a fragmented HA results in activation of MAP kinase and, subsequently, enhances uPA and uPAR expression and facilitates invasion of human chondrosarcoma cells.

Apparent trans-chromosomal antibody class switch in mice bearing an igh a μ-chain transgene on an igh b genetic background

Native high molecular weight dextran induces a thymus-independent response in BALB/c mice. When the dextran epitope is linked to a protein carrier the response becomes thymus-dependent. IgG antibodies produced after secondary immunization had epitope specificity and idiotope of myeloma M104E. The antibody of M104E (μ,λ1) is representative for antibodies produced by mice with immunoglobulin haplotype Igh a in response to immunization with dextran B1355S. Myeloma product and physiological antibodies share specificity for the α(1–3) glucosidic linkage and have idiotopes in common. Mice with haplotypes other than Igh a (e.g. Igh b) are unable to yield this type of response. A complete rearranged immunoglobulin μ-chain gene with a VDJ-region from BALB/c (Igh a) myeloma protein M104E had been introduced into the genome of BALB/c congenic mice having the haplotype Igh b. As was shown previously in our laboratory the M104E p-chain transgene confers Igh a-type reactivity to Igh b Mice. In experiments described in this report we used the thymus-dependent form of the antigen to immunize mice bearing the M104E λ 1-chain, either alone or together with the λ 1-chain, as a transgene on an Igh b genetic background. Serological analysis revealed a class switch to IgG very similar to that seen in BALB/c mice with respect to magnitude, kinetics, epitope and idiotope specificity. The pattern of IgG subclass expression was indistinguishable in p-chain transgenic Igh b and normal BALB/c mice. The class switch occurred even though, as is shown here, the transgene had become incorporated in a site not linked to the Igh locus on chromosome 12. We propose a model for this apparent trans-chromosomal class switch recombination which is based on mechanisms known for conventional switch recombination.

Heterologous expression of a wheat high molecular weight glutenin gene in Escherichia coli.

An engineered DNA fragment containing a DNA sequence encoding a wheat high molecular weight glutenin subunit was cloned into a bacterial expression vector that is based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the wheat high molecular weight glutenin subunit in Escherichia coli. This protein comigrated in SDS/PAGE with a native high molecular weight glutenin subunit extracted from wheat endosperm and cross-reacted with antibodies raised against purified wheat high molecular weight glutenins. The wheat subunit synthesized in E. coli also exhibited pI and solubility characteristics identical to those of native wheat subunits. Moreover, the wheat glutenin subunit produced in E. coli cells self-assembled into oligomers linked by intermolecular disulfide bonds, a process that plays an important role in the assembly of native glutenins during gluten formation. The large amounts of a purified wheat subunit obtained from E. coli will enable investigators to analyze the three-dimensional structure of that protein and to identify protein sequences that affect the bread-making quality of wheat dough.

Recent advances in the study of biopolymers

AbstractIn describing some of the macromolecular characteristics of the nucleic acids, particularly DNA, and of representative proteins, the following aspects are discussed: computer‐assisted modeling as a useful tool for depicting the three‐dimensional structures and macromolecular characteristics of biopolymers; conformational flexibility in biopolymers; parameters determining protein recognition; and the importance of the three‐dimensional structure of antibodies, enzymes, receptors, ion channels, and proteins interacting with nucleic acids, in determining their characteristic biological functions. Many of the problems raised by molecular biologists concerning the mode of action of biopolymers could be tackled by polymer chemists, because of their experience in dealing theoretically as well as experimentally with synthetic and native high molecular weight compounds.