Microvillus Membrane Research Articles

Large Intravenous Loads of Bilirubin Photoconversion Products, in Contrast to Bilirubin, Do Not Cause Cholestasis in Bile Acid-Depleted Pigs

Background: Large intravenous bilirubin infusions in bile acid-depicted pigs (BADF) destroy hepatocyte canalicular membrane microvilli (CMV) and cause cholestasis. This study examines whether bilirubin photoconversion product infusions do the same. Methods: The effects of systemic infusion of 135 µmol - kg−1 body weight bilirubin photoconversion products on CMV density and cholcretic response to intruportal bile acid infusion were studied in BADP. Furthermore, the effects of 135 µmol - kg−1 b.w. bilirubin infusion, cither through an artcriovenous bilirubin photoconversion shunt device (PCD) or intravenously, were measured in PCD-connected BADP. Results: Intravenous bilirubin photoconversion product infusions affected neither the CMV density nor the choleretic response to cholic acid infusion, and neither did bilirubin infusion through the PCD. In contrast, intravenous bilirubin infusion caused canalicular injury and cholestasis in four of six PCD-connected 13ADP. Conclusion: Bilirubin photo-conversion products do not destroy CMV or cause cholestasis in BADP. A bilirubin photoconversion shunt device can confer cholestasis protection to bilirubin-loaded BADP.

Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (hemiptera: pyrrhocoridae) midgut cells

The midgut of Dysdercus peruvianus is divided into four sections (V 1-V 4). All the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V 1 and V 2 tissue in isotonic and hypotonic conditions showed that α-glucosidase is associated with membranous structures larger than those associated with β-glucosidase. The α/β-glucosidase activity ratio is 34 ± 4 in V 1 tissue and 170 ± 10 in membranes recovered from the V 1 luminal contents. These membranes are resolved in sucrose gradients into low density (1.087 ± 0.001 g/ cm 3) α-glucosidase-carrying membranes (α/β-glucosidase activity ratio of 330±30) and high density (1.132 ± 0.002 g/ cm 3) β-glucosidase-carrying-membranes. Low-density membranes have 1090 ± 60 μg lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V 1 luminal contents are composed mainly of a-glucosidase-rich membranes. The data suggest that α-glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. β-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes.

Proteolytic Processing of Human Lactase-Phlorizin Hydrolase Is a Two-Step Event: Identification of the Cleavage Sites

Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the cleavage site at Arg734-Leu735. Treatment of pro-lactase-phlorizin hydrolase expressed in COS-1 and MDCK cells by trypsin yielded a 145-kDa form with an identical amino terminal as the mature microvillus enzyme isolated from intestinal mucosa (Ala869). These data provide unambiguous evidence of a two-step processing of human lactase-phlorizin hydrolase. The first cleavage occurs intracellularly after a dibasic site (Arg734-Leu735) and yields the 160-kDa intermediate form. In a second step the intermediate form inserted into the microvillus membrane is trimmed to the mature enzyme by luminal trypsin.

The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes.

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.

Metabolism of vasopressin, oxytocin and their analogues [Mpa1, d-Arg8]-vasopressin (dDAVP) and [Mpa1, d-Tyr(Et)2, Thr4, Orn8]-oxytocin (antocin) in human kidney and liver homogenates

Information regarding the metabolic fate of the neurohypophyseal hormones arginine-vasopressin (AVP), oxytocin (OT) and their analogues in man is practically non-existent. The aim of the present study was to investigate the stability of oxytocin, vasopressin and their analogues dDAVP and [Mpa1-d-Tyr2(Et), Thr4, Orn8]-oxytocin (antocin) in human renal microvilli brush border membranes and in human liver membranes. After incubation the extent of degradation of the peptides was determined by reversed phase high-performance liquid chromatography (HPLC). The degradation of both AVP and OT was rapid in the presence of glutathione and human renal microvilli membranes. AVP, as well as dDAVP, was stable when incubated with microvilli membranes without glutathione, while OT was metabolized. The metabolization of the oxytocin analogue, antocin, also varied with the presence of glutathione. While in the absence of glutathione a more lipophilic peak eluted, a more hydrophilic peak was observed with glutathione on HPLC. The lipophilic peak was found to coelute with the truncated analogue [Mpa1, d-Tyr2 (Et), Thr4, desOrn8, Gly9]-oxytocin. No degradation occurred when the peptides were incubated with liver membranes. However, when using crude, unpurified liver homogenate degradation occurred for all peptides except antocin. The degradation of AVP in the human unpurified liver homogenate was as rapid as in the renal microvilli membranes. Similarly, OT was more rapidly degraded in human kidney microvilli membranes in the presence of glutathione than in the human crude liver homogenate, when using equal amounts of protein in the incubations. Thus, the present investigation indicates the existence of two possible metabolic pathways, in kidney microvilli, one for OT, which did not require the presence of reduced glutathione, and one for AVP, which required the presence of reduced glutathione. Liver degradation, on the other hand, requires the hepatocytes.

Changes in the concentration of microvilli on the free surface of healing mesothelium are associated with alterations in surface membrane charge.

The luminal plasmalemma of regenerating mesothelial cells was examined by transmission electron microscopy and the concentration of microvilli at various stages of healing was quantified. Charged tracer and lectin binding techniques were also employed to investigate electrostatic and chemical changes in mesothelial glycocalyx. In uninjured mesothelium and at all stages of healing, the concentration of microvilli at the cellular periphery was greater than over the main cell mass (P < 0.05). Furthermore, there was an increase in the concentration of microvilli in all regions by day 4, which reached a maximum at day 6, then at days 10-15 returned to values closer to uninjured mesothelium (P < 0.01). These changes were associated with an alteration in surface charge. In all lesions, the surface charge on microvillar membranes was greater than for flat membranes, but the difference was only significant at days 4, 6, and 15 (P < 0.001). The changes in surface charge may reflect a differential expression of mucopolysaccharides on the surface membrane. In addition, concanavalin A bound avidly to mesothelial surface membranes, suggesting the presence of alpha-methyl-D-mannoside residues. These findings suggest an association between microvillar formation and surface charge, the former protecting the healing mesothelium by enhancing entrapment of serosal fluid and its contents.

CHANGES IN THE CONCENTRATION OF MICROVILLI ON THE FREE SURFACE OF HEALING MESOTHELIUM ARE ASSOCIATED WITH ALTERATIONS IN SURFACE MEMBRANE CHARGE

The luminal plasmalemma of regenerating mesothelial cells was examined by transmission electron microscopy and the concentration of microvilli at various stages of healing was quantified. Charged tracer and lectin binding techniques were also employed to investigate electrostatic and chemical changes in mesothelial glycocalyx. In uninjured mesothelium and at all stages of healing, the concentration of microvilli at the cellular periphery was greater than over the main cell mass (P<0·05). Furthermore, there was an increase in the concentration of microvilli in all regions by day 4, which reached a maximum at day 6, then at days 10–15 returned to values closer to uninjured mesothelium (P<0·01). These changes were associated with an alteration in surface charge. In all lesions, the surface charge on microvillar membranes was greater than for flat membranes, but the difference was only significant at days 4, 6, and 15 (P<0·001). The changes in surface charge may reflect a differential expression of mucopolysaccharides on the surface membrane. In addition, concanavalin A bound avidly to mesothelial surface membranes, suggesting the presence of α-methyl-d-mannoside residues. These findings suggest an association between microvillar formation and surface charge, the former protecting the healing mesothelium by enhancing entrapment of serosal fluid and its contents.

Interactions ofBacillus thuringiensisCrystal Proteins with the Midgut Epithelial Cells ofSpodoptera frugiperda(Lepidoptera: Noctuidae)

Binding of differentBacillus thuringiensisinsecticidal crystal proteins (ICPs) to the midgut epithelium ofSpodoptera frugiperdalarvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. Our results show that ICPs interact with the microvilli of epithelial cells ofS. frugiperdain two different ways. The first is typical of highly toxic proteins (like CryIC and CryID); this interaction is saturable and specific. In contrast, some nontoxic proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor. The CryIC toxin binds to two brush border proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa. Immunological detection of ingestedB. thuringiensisICPs on gut sections ofS. frugiperdalarvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane. Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs alsoin vivoalthough it is not related to toxicity.

Latencies of calcium elevation and depolarization in Limulus ventral photoreceptors injected with GDP-βS

We have used confocal fluorescence microscopy and fluorescent calcium indicators to investigate the relationship between light-induced elevation of intracellular calcium ion concentration (Ca i) and depolarization in small volumes of cytosol close to the microvillar plasma membrane of the ventral photoreceptor of Limulus polyphemus. We prolonged the latency of the light response by treatment of cells with hydroxylamine and injection of the G-protein blocker, guanosine-5′-O-(2-thiodiphosphate (GDP-βS). Such treatment increased the latency of the cell's response from approximately 20 to 50 ms. In both treated and untreated cells we observed a close correlation between the times at which we first detected the electrical response and the elevation of Ca i. We obtained 18 out of 54 and 12 out of 22 recordings, in untreated and treated cells respectively, for which the elevation of Ca i was detected simultaneous with, or 1–4 ms prior to, the electrical response to light. The prolonged latent period exhibited by treated cells may make possible future investigation of the effects on the initial response to light of local photolytic release of caged compounds at the microvillar membrane.

Mechanism of H(+)-coupled formate transport in rabbit renal microvillus membranes.

It has been proposed that a major fraction of Cl- absorption in the mammalian proximal tubule occurs by Cl-/formate exchange across the apical membrane with recycling of formate by nonionic diffusion. The purpose of this study was to characterize the mechanism of formate recycling in rabbit renal microvillus membrane vesicles. Formate uptake was stimulated by an inside-alkaline pH gradient. When external pH (pH alpha) was varied at constant internal pH (pHi), the initial rate of formate uptake was less than predicted for nonionic diffusion of formic acid at constant formic acid permeability. When pHi was varied at constant pHi, the initial rate of formate uptake exhibited cooperative and saturable kinetics with respect to pHi, in contrast to the pHi independence predicted for nonionic diffusion. pH gradient-stimulated [14C]formate uptake was stimulated by internal formate, indicating formate/formate exchange. pH gradient-stimulated formate influx was sensitive to inhibition by 1 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid but not by furosemide or hydroxycinnamate. We conclude that pH gradient-stimulated formate uptake takes place by a carrier-mediated process of H(+)-formate cotransport, OH-/formate exchange, or facilitated formic acid diffusion, rather than solely by passive nonionic diffusion through the lipid bilayer.

Changes in cellular ultrastructure induced by gamma-interferon in K562 cells may be prerequisite for apoptosis.

We report here the time-course of electron microscopic changes induced by gamma-interferon (IFN-gamma) in the human erythromyeloid leukemia cell line K562. In K562 cells treated with IFN-gamma for 6h, the nuclei were polygonal in shape and microvilli were far more abundant on cell membranes compared with control K562 cells, and invaginations were often seen in the cell membranes. There was a reduction in the number of cell-membrane microvilli and an increase in the number of lysosomal bodies in the cytoplasm of K562 cells treated with IFN-gamma for 12h. After treatment with IFN-gamma for 24h, the cell membrane microvilli disappeared, large numbers of cellular organelles were observed, such as mitochondria and lysosomes, and the cytoplasm became electron-dense. Cytoplasmic vesicles and vacuoles were also observed. These vesicles may correspond to an intermediate step in the ultimate cellular disintegration associated with apoptosis caused by IFN-gamma.

Characterization of IGF-I receptors in the porcine small intestine during postnatal development

Receptors for insulin-like growth factor I were characterized on intestinal membranes of jejunum from newborn, suckling, and weaned pigs. 125I-labelled insulin-like growth factor I (IGF-I) binding was time-dependent, saturable, linearly related to membrane protein, and reversible. Analysis of saturation curve data from membrane homogenates revealed a single class of receptors, with receptor number declining during development. In vitro autoradiography localized receptors to the small intestinal submucosa and mucosa and further immunohistochemical localization identified receptors on the apical (microvillar membrane) and basolateral surfaces of enterocytes. After affinity cross-linking of 125I-labelled IGF-I to microvillar membranes, 135 and 260 kilodalton bands were visualized on polyacrylamide gels under reducing conditions. Parallel incubations with excess unlabeled IGF-I, -II, or insulin demonstrated that these bands correspond to the α subunit and an incompletely reduced dimeric α complex of the IGF-I receptor. Receptor number on microvillar membranes increased dramatically during the suckling period and was negatively correlated with maltase and sucrase activities. The functional activity of receptors on microvillar membranes was demonstrated using an in vitro phosphorylation assay. The expression of functional IGF-I receptors during development on both apical and basolateral membranes suggests a regulatory role for this receptor and its ligands.

P-17-5 - Regional cerebral perfusion by Tc-99m HMPAO SPECT before and after tacrine in alzheimer's disease

Microvillus membrane vesicles isolated from renal cortex have subsequently served as a useful model system for characterizing the kinetic and biochemical features of a Na+–H+ exchanger whose properties are largely representative of plasma membrane Na+–H+ exchangers in general. This chapter reviews studies on the molecular properties of the renal microvillus membrane Na+–H+ exchanger. Additional transport systems for the transfer of acids or bases exist in parallel with the Na+–H+ exchanger on the luminal membrane of proximal tubule cells. The chapter describes the kinetics of the renal Na+–H+ exchanger. As the imidazolium ring of histidine is the principal ionic group in proteins that is titratable at near-neutral pH values, in the study described in the chapter, this helped to evaluate the effect of histidine-specific reagents on the activity of the renal microvillus membrane Na+–H+ exchanger. The carboxyl-activating reagent N-ethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline (EEDQ) inhibits Na+–H + exchange in renal microvillus vesicles. Also, The observations that N,N'-dicyclohexyl-carbodiimide (DCCD) inactivates the renal Na+–H+ exchanger irreversibly and that amiloride protects against this inactivation suggested a strategy for covalently labeling the transport protein or one of its subunits. The renal Na+–H+ exchanger helps in facilitating anion transport by HCO−3 reabsorption, organic anion reabsorption, Cl− reabsorption, and anion transport and maintaining intracellular pH.

Pathways for Oxalate Transport in Rabbit Renal Microvillus Membrane Vesicles

Recent evidence suggests that apical membrane Cl--oxalate exchange plays a major role in mediating Cl- absorption in the renal proximal tubule. To sustain steady-state Cl- absorption by a mechanism of exchange for intracellular oxalate requires the presence of one or more pathways for recycling oxalate from lumen to cell. Accordingly, we evaluated the mechanisms of oxalate transport in luminal membrane vesicles isolated from the rabbit renal cortex. We found that transport of oxalate by Na+ cotransport is negligible compared to the transport of sulfate. In contrast, we demonstrated that oxalate shares the electroneutral pathway mediating Na+-independent sulfate-carbonate exchange. We also demonstrated the presence of OH--oxalate exchange (indistinguishable from H+-oxalate cotransport). The process of OH--oxalate exchange was electrogenic and partially inhibited by Cl-, indicating that it occurs, at least in part, as a mode of the Cl--oxalate exchanger described previously. An additional component of OH--oxalate exchange was insensitive to inhibition by either Cl- or sulfate, suggesting that it takes place by neither the Cl--oxalate exchanger nor the sulfate-carbonate exchanger. We conclude that multiple anion exchange mechanisms exist by which oxalate can recycle from lumen to cell to sustain Cl- absorption occurring via apical membrane Cl--oxalate exchange in the renal proximal tubule.

Interaction of verotoxin 2e with pig intestine

In pigs with edema disease, verotoxin 2e (VT2e) is produced in the intestine and transported to tissues, but neither the mechanism by which toxin passes through the intestine nor its failure to induce an enterotoxic reaction is understood. Binding of VT2e to pig intestine was examined by enzyme-linked immunosorbent assay involving microvillus membranes (MVM) and crude mucus; thin-layer chromatographic overlay immunoassay with total lipids extracted from MVM; and indirect immunofluorescence of toxin bound to thin sections of jejunum, ileum, and colon. VT2e bound significantly to MVM from pig jejunum and ileum but not to crude mucus. Verotoxin 2e-binding glycolipids, globotetraosylceramide and globotriaosylceramide, were detected by thin-layer chromatographic overlay immunoassay in extracts of MVM from jejunum and ileum. Indirect immunofluorescence showed that VT2e bound to vessels within the submucosa and muscularis mucosa of the jejunum, ileum, and colon and to enterocytes at the lower portion but not at the tips of villi in the jejunum and ileum. Receptors for VT2e are therefore present in the intestine of the pig, but their role in absorption of VT2e is unclear since intraintestinal inoculation of pigs with large quantities of VT2e does not result in edema disease. Previously reported lack of enterotoxicity of verotoxins in pig intestine may be explained by the absence of toxin receptors in the villus absorptive enterocytes.

ATP-diphosphohydrolase activity in rat renal microvillar membranes and vascular tissue

Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations. The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney. ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to ‘right side out’ membranes, were characterized. The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney. ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties. These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap 5A. The M r of native ATPase and ADPase activities was determined by the 60Co irradiation—inactivation technique being around 65 kDa for both hydrolytic activities. Immunowestern blot analysis also supports the presence of apyrase in microvilli. Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Rat intestinal mucosa contains high diacylglycerol-synthesizing activity (monoacylglycerol acyltransferase (MGAT) activity) due to monoacylglycerol and fatty acid, independently of coenzyme A and ATP. MGAT activity was purified from rat intestinal mucosa by successive chromatography separations on DEAE-cellulose, CM- Sephadex, and anti-IgG-Sepharose against rat pancreatic lipase. The enzyme was electrophoretically homogeneous, and its molecular weight was 49,000, which is identical with that of rat pancreatic lipase. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000. The activity of the purified enzyme was completely inhibited by addition of the antibody. Using immunocytochemical techniques, it was found that immunoreactive protein against rat pancreatic lipase was uniformly distributed within the absorptive cells of the intestine but was absent from the microvillar membrane. The MGAT activity of intestinal mucosal homogenate was inhibited by about 65% by addition of antibody against rat pancreatic lipase. Trioleoylglycerol- and dioleoylglycerol-hydrolyzing activities of the purified enzyme and pancreatic lipase were inhibited by addition of intestinal mucosa extract. These results suggest that pancreatic lipase is present in intestinal absorptive cells and that it may contribute to resynthesis of diacylglycerol from monoacylglycerol and fatty acids in these cells.

Disaccharide Digestion and Maldigestion

All food carbohydrates are hydrolysed to monosaccharides before transport across the microvillus membrane. The digestion of disaccharides and some oligosaccharides is undertaken by a number of small intestinal brush border enzymes: sucrase-isomaltase, lactase phlorizinhydrolase, maltase-glycoamylase and trehalase. The distribution of the enzymes in the small intestine has been investigated. Different disaccharide maldigestion syndromes have been described. Lactase deficiency in adults is a condition found in the majority of inhabitants of the world. However, the prevalence varies widely between different populations. Sucrase-isomaltase deficiency is a very rare congenital condition except in Greenland. Trehalose maldigestion is likewise rare outside Greenland. Different hypotheses regarding the molecular background of the maldigestion syndromes are discussed.

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments.

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.