Evaluation of structural and secretory glycoconjugates in normal human jejunum by means of lectin histochemistry

Summary The labelling pattern of eight lectins was studied in jejunal samples from ten normal subjects, in order to define the normal distribution of structural and secretory glycoconjugates in the small bowel.The following lectins were studied by means of a peroxidase technique on formalin-fixed samples: Arachis hypogaea, Ricinus communis, Canavalia ensiformis, Lens culinaris, Phaseolus vulgaris, Triticum vulgaris, Ulex europaeus, Dolichos biflorus. Phaseolus vulgaris reacted with goblet cell mucus throughout the villus-crypt axis.Conversely Ulex europaeus, Dolichos biflorus and Triticum vulgaris lectin labelling of globet cells appeared to be confined to the upper part of the villi. This finding suggests that during cell migration from crypt to villus tip, the continuing maturation of goblet cells is associated with the differentiation of secretory carbohydrates, which probably parallels the cell maturation cycle. Lectin histochemistry appears to be a reliable tool for the study of structural and secretory glycoconjugates in the jejunal mucosa, and might be of value in the study of diseases in which the cell-maturation cycle in the small bowel is altered.     

Evaluation of structural and secretory glycoconjugates in normal human jejunum by means of lectin histochemistry

The labelling pattern of eight lectins was studied in jejunal samples from ten normal subjects, in order to define the normal distribution of structural and secretory glycoconjugates in the small bowel. The following lectins were studied by means of a peroxidase technique on formalin-fixed samples: Arachis hypogaea, Ricinus communis, Canavalia ensiformis, Lens culinaris, Phaseolus vulgaris, Triticum vulgaris, Ulex europaeus, Dolichos biflorus. Phaseolus vulgaris reacted with goblet cell mucus throughout the villus-crypt axis. Conversely Ulex europaeus, Dolichos biflorus and Triticum vulgaris lectin labelling of goblet cells appeared to be confined to the upper part of the villi. This finding suggests that during cell migration from crypt to villus tip, the continuing maturation of goblet cells is associated with the differentiation of secretory carbohydrates, which probably parallels the cell maturation cycle. Lectin histochemistry appears to be a reliable tool for the study of structural and secretory glycoconjugates in the jejunal mucosa, and might be of value in the study of diseases in which the cell-maturation cycle in the small bowel is altered.     

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = −440 mV, is lower than that of Fd1, Em = −372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP⁺ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV–Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys₅₃ValAsnCys₅₆ of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.     

Apoptosis in the monkey small intestinal epithelium: structural and functional alterations in the mitochondria

Our earlier studies have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. Because mitochondria have been implicated in the apoptotic process, this study looked at the function and lipid composition of mitochondria isolated from apoptotic villus tip cells and compared it with middle and crypt cells. Decreased MTT reduction and respiratory control ratio, increased swelling and altered mitochondrial enzyme activities were seen in the villus tip cell mitochondria when compared to other cells. The lipid composition of the villus tip mitochondria were different from the other mitochondria. A decrease in phosphatidylethanolamine and phosphatidyl-inositol and an increase in phosphatidic acid was seen in these mitochondria. Fatty acid composition analysis showed more unsaturated fatty acids in the free fatty acid and phospholipid fraction in villus tip cell mitochondria as compared to other cells. These studies suggest that in the monkey small intestinal epithelium, apoptotic process is associated with functional and structural alterations in the mitochondria.     

Effect of trifluoroethanol on the structural and functional properties of alpha-crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of - and -crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native -crystallin and its two constituent subunits. A conformational change occurs from the characteristic -sheet to the -helix structure in both native -crystallin and its subunits with the increase in TFE levels. Among the two subunits, A-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of B-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of A-crystallin to bind and prevent the aggregation of B-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of A-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of A-crystallin—to stabilize B-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers

We provide here new insights into rotavirus (RRV) pathogenicity by showing that RRV infection promotes structural and functional injuries localized at the tight junctions (TJ) in the cell-cell junctional complex of cultured polarized human intestinal Caco-2 cells forming monolayers. RRV infection resulted in a progressive increase in the paracellular permeability to [(3)H]mannitol as a function of the time postinfection. We observed a disorganization of the TJ-associated protein occludin as a function of the time postinfection, whereas distribution of the zonula adherens associated E-cadherin was not affected. These structural and functional RRV-induced TJ injuries were not accompanied by alteration in cell and monolayer integrity, as assessed by the lack of change in transepithelial membrane resistance and lactate dehydrogenase release. Finally, using the stabilizer of actin filaments Jasplakinolide, we demonstrated that the RRV-induced structural and functional alterations in TJ are independent of the RRV-induced apical F-actin rearrangements.     

Homocysteine modifies structural and functional properties of fibronectin and interferes with the fibronectin-fibrillin-1 interaction

Homocystinuria is a genetic disorder resulting in elevated levels of homocysteine in plasma and tissues. Some of the skeletal and ocular symptoms such as long bone overgrowth, scoliosis, and ectopia lentis overlap with symptoms seen in Marfan syndrome. Marfan syndrome is caused by mutations in the extracellular matrix protein fibrillin-1. We previously showed that fibrillin-1 is a target for homocysteine and that the deposition of homocysteinylated fibrillin-1 in the extracellular matrix is compromised. Since the assembly of fibrillin-1 is critically dependent on fibronectin, we analyzed the consequences of fibronectin homocysteinylation and its interaction with fibrillin-1. Cellular fibronectin and proteolytic fragments were homocysteinylated and tested in various interaction assays with recombinant fibrillin-1 and heparin. Fibronectin homocysteinylation consistently compromised the fibronectin-fibrillin-1 interaction, while the interaction with heparin was not affected. Fibronectin homocysteinylation, but not cysteinylation, reduced the fibronectin dimers to monomers as shown by Western blotting. ELISA analyses of homocysteinylated fibronectin with three monoclonal antibodies demonstrated structural changes in the disulfide-containing FNI domains FNI(2), FNI(4), and FNI(9). Using fluorescently labeled fibronectin, we studied the consequence of fibronectin homocysteinylation on assembly in cell culture. Modified fibronectin showed deficiencies in denovo matrix incorporation and initial assembly. In conclusion, we define here characteristic structural changes of fibronectin upon homocysteinylation that translate into functional deficiencies in the fibronectin-fibrillin-1 interaction and in fibronectin assembly. Since fibronectin is a major organizer of various extracellular protein networks, these structural and functional alterations may contribute to the pathogenesis of homocystinuria and Marfan syndrome.     

Electrical coupling in the circular muscles of dog jejunum.

Two distinct layers of circular muscle have previously been demonstrated in dog jejunum, the main circular layer containing many gap junction contacts, and an inner dense muscle layer where no gap junctions have been found. Length constants were determined for these muscle layers and no significant difference was found between these values. The main circular muscle cells had lower membrane potentials and may have had abnormally low space constants owing to injury. It was concluded that the absence of gap junctions in the inner dense layer does not reduce the spread of passive current as might be expected of electrically isolated cells, and it is suggested that an alternate pathway for passive current exists in this layer.     

Isolation and some structural and functional properties of macrophage tropomyosin

Tropomyosin purified from rabbit lung macrophages is very similar in structure to other nonmuscle cell tropomyosins. Reduced and denatured, the protein has two polypeptides which migrate during electrophoresis in sodium dodecyl sulfate on polyacrylamide gels with slightly different mobilities corresponding to apparent Mr's of about 30 000. Following cross-linking by air oxidation in the presence of CuCl2, electrophoresis under nonreducing conditions reveals a single polypeptide of Mr 60 000. Macrophage tropomyosin has an isoelectric point of 4.6 and an amino acid composition similar to other tropomyosins. It contains one cysteine residue per chain. In the electron microscope, macrophage tropomyosin molecules rotary shadowed with platinum and carbon are slender, straight rods, 33 nm in length. Macrophage tropomyosin paracrystals grown in high magnesium concentrations have an axial periodicity of 34 nm. On the basis of yields from purification and from two-dimensional electrophoretic analyses of macrophage extracts, tropomyosin comprises less than 0.2% of the total macrophage protein, a molar ratio of approximately 1 tropomyosin molecule to 75 actin monomers in the cell. Macrophage tropomyosin binds to actin filaments. Macrophage, skeletal muscle, and other nonmuscle cell tropomyosins inhibit the fragmentation of actin filaments by the Ca2+-gelsolin complex. The finding implies that tropomyosin may have a role in stabilizing actin filaments in vivo.     

A novel approach to studying the structural and functional properties of proteins with unknown functions

Three proteins from extremophilic bacteria—hypothetical monooxygenase from Deinococcus radiodurans, hypothetical nucleotidyl transferase from Thermotoga maritime, and hypothetical oxidoreductase from Exiguobacterium sibiricum—and the DJ-1 chaperone protein from Homo sapiens have been produced in Escherichia coli. The isolation and purification procedures developed for the recombinant proteins allowed us to achieve yields higher than 96%. Crystallization conditions enabling stable growth of crystals have been determined. X-ray experiments have been performed to test the quality of the crystals and the resolution achieved ranged from 1.2 to 1.8 Å.     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Gel filtration and SDS-PAGE separation of hemoglobin (Hb) irradiated under argon or N2O show formation of covalent-aggregated Hb molecules. The production of covalent bonds is attributed mainly to the action of hydroxyl radicals, because addition of ethanol, a scavenger of these radicals, suppresses this reaction to a great extent. The oxidized heme iron forming metHb or hemichromes is found in all the separated fractions of irradiated Hb. It is also found that the radiation-modified Hb molecules exhibit a decrease of co-operative binding of oxygen.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.     

Deamidation affects structural and functional properties of human alphaA-crystallin and its oligomerization with alphaB-crystallin

To determine the effects of deamidation on structural and functional properties of alphaA-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated alphaB-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206-214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of alphaA mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of alpha-crystallin containing (i) wild type alphaA (WT-alphaA): WT-alphaB crystallins, (ii) individual alphaA-deamidated mutants:WT-alphaB crystallins, and (iii) WT-alphaA:individual alphaB-deamidated mutant crystallins. Compared with the WT-alphaA, the three alphaA-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-alphaA and WT-alphaB, the heteroaggregates containing deamidated subunits of either alphaA- or alphaB-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-alphaA and deamidated alphaB subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated alphaA- and WT-alphaB subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of alphaA-crystallin and (b) the presence of WT-alphaB in the alpha-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.     

Effect of cAMP on the structural and functional properties of the erythrocytes]

Cyclic AMP inhibits the anion transport and decreases the osmotic resistance and deformability of erythrocytes with the normal level of ATP. With ATP-depleted erythrocytes cAMP exerted the opposite effects on the corresponding characteristics. In addition, it was observed that the pattern of cAMP effect on the cell form depends on the basal level of ATP. These effects may be associated with the two types of structural rearrangements of erythrocyte membranes established earlier: a cooperative transition not connected with protein kinase system, and a non-cooperative one caused by protein phosphorilation.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.