The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts.

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.

Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms.

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells.

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.

The mouse Ptprr gene encodes two protein tyrosine phosphatases, PTP-SL and PTPBR7, that display distinct patterns of expression during neural development.

The protein tyrosine phosphatases PTP-SL and PTPBR7 differ only in the length of their N-terminal domain. We show here that PTP-SL and PTPBR7 are isoforms derived from a single gene (Ptprr) through developmentally regulated use of alternative promoters. Isoform-specific reverse transcriptase-polymer chain reaction (RT-PCR) and RNA in situ hybridization experiments reveal that PTPBR7 is expressed during early embryogenesis in spinal ganglia cells as well as in developing Purkinje cells. Post-natally, PTPBR7 is expressed in various regions of the adult mouse brain, but expression in Purkinje cells has ceased and is replaced by the PTP-SL-specific transcript. In transient transfection experiments it is confirmed that PTPBR7 is a type I transmembrane protein tyrosine phosphatase (PTPase). PTP-SL, however, appears to be a cytosolic membrane-associated PTPase that is located at perinuclear vesicular structures that partly belong to the endosomal compartment. Thus, during maturation of Purkinje cells, a gene-promoter switch results in the replacement of a receptor-type PTPase by a cytosolic vesicle-associated isoform.

Effects of anabolic steroids on diaphragm impairment induced by methylprednisolone in emphysematous hamsters.

This study was designed to investigate whether the administration of the anabolic steroid nandrolone decanoate is able to antagonize the loss in diaphragm function induced by long-term administration of a low-dose of methylprednisolone in emphysematous hamsters. Normal and emphysematous male hamsters were randomized to receive either saline or methylprednisolone 0.2 mg x kg(-1) x day(-1) for 9 months, with or without nandrolone decanoate 1 mg x kg(-1) x week(-1) i.m. during the final 3 months. Diaphragm contractile properties and myosin heavy chain composition were determined. Compared to control hamsters, the force generating capacity of isolated diaphragm strips decreased by approximately 12% in the emphysema group and by approximately 22% in the emphysema plus methylprednisolone group. Addition of nandrolone decanoate to the emphysema plus methylprednisolone hamsters significantly improved force generation. The atrophy of type IIa and IIx diaphragm fibres in the emphysema plus methylprednisolone group was completely reversed to the level of control hamsters by the addition of nandrolone decanoate. In conclusion, nandrolone decanoate in part reversed the loss in diaphragm force-generating capacity in emphysematous hamsters treated with methylprednisolone, and reversed type IIa and IIx fibre atrophy completely.

Analysis of a naturally occurring mutation in sucrase-isomaltase: glutamine 1098 is not essential for transport to the surface of COS-1 cells.

A glutamine for proline substitution at position 1098 was previously shown to result in accumulation of brush-border sucrase-isomaltase in the Golgi apparatus. The substitution is present in a highly homologous region of the protein, and results in a comparable accumulation when introduced into the same region in lysosomal alpha-glucosidase. To study the importance of the glutamine-1098, we analyzed the transport compatibility of two mutants in which glutamine-1098 is substituted by lysine or alanine. Both mutants were transported to the cell surface and processed comparable to wild type. We concluded that glutamine-1098 is not essential for transport to the cell surface.

Anabolic steroids in part reverse glucocorticoid-induced alterations in rat diaphragm.

Animal and clinical studies have shown respiratory muscle dysfunction caused by treatment with glucocorticoids. The present study was designed to investigate whether anabolic steroids are able to antagonize the loss of diaphragm force induced by long-term low-dose methylprednisolone (MP) administration. Male adult rats were randomized to receive saline or MP (0.2 mg . kg-1 .day-1 sc) during 9 mo, with or without nandrolone decanoate (ND; 1 mg . kg-1 . wm -1 im) during the last 3 mo. The approximately 10% reduction in force generation of isolated diaphragm bundles induced by MP was completely abolished by addition of ND. The MP-induced decrease in number of fibers expressing type IIb myosin heavy chains was not reversed by ND. MP slightly reduced type I, IIa, and IIx fiber cross-sectional areas (CSA), but not type IIb fiber CSA. Addition of ND abolished the reduction in IIa and IIx fiber CSA. The MP-induced alterations in glycogenolytic activity and fatty acid oxidation capacity were not reversed by ND. In conclusion, the marked reduction in diaphragm force caused by long-term low-dose MP was completely abolished by addition of ND. ND in part also antagonized the effects of MP on diaphragm morphology but showed no beneficial effects on biochemical changes.

Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.

Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.

Impaired mammary gland development and function in mice lacking LAR receptor-like tyrosine phosphatase activity.

The LAR receptor-like protein tyrosine phosphatase is composed of two intracellular tyrosine phosphatase domains and a cell adhesion molecule-like extracellular region containing three immunoglubulin-like domains in combination with eight fibronectin type-III-like repeats. This architecture suggests that LAR may function in cellular signalling by the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. We used gene targeting in mouse embryonic stem cells to generate mice lacking sequences encoding both LAR phosphatase domains. Northern blot analysis of various tissues revealed the presence of a truncated LAR mRNA lacking the cytoplasmic tyrosine phosphatase domains and indicated that this LAR mutation is not accompanied by obvious changes in the expression levels of one of the LAR-like receptor tyrosine phosphatases PTPdelta or PTPsigma. LAR-/- mice develop and grow normally and display no appreciable histological tissue abnormalities. However, upon breeding we observed an abnormal neonatal death rate for pups from LAR-/- females. Mammary glands of LAR-/- females were incapable of delivering milk due to an impaired terminal differentiation of alveoli at late pregnancy. As a result, the glands failed to switch to a lactational state and showed a rapid involution postpartum. In wild-type mice, LAR expression is regulated during pregnancy reaching maximum levels around Day 16 of gestation. Taken together, these findings suggest an important role for LAR-mediated signalling in mammary gland development and function.

Effects of long-term low-dose methylprednisolone on rat diaphragm function and structure.

In animal studies, high dosages of corticosteroids cause changes in diaphragm structure and function. The present study was designed to investigate the effects of long-term low-dose methylprednisolone (MP) administration on rat diaphragm contractile properties and morphology. Thirty adult rats were treated with saline or MP (0.2 mg/kg/day s.c.) during 6 months. Contractile properties of isolated diaphragm strips, immunohistochemical characteristics analyzed by means of antibodies reactive with myosin heavy chain isoforms, and enzyme activities were determined in the diaphragm muscle. MP significantly reduced diaphragm force generation by -15% over a wide range of stimulation frequencies. The number of type IIb fibers was reduced by MP. There was a mild but significant decrease in type I and IIa fiber cross-sectional area (CSA), whereas type IIx and IIb CSA did not change. These changes resulted in a reduction in the relative contribution of type IIb fibers to total diaphragm muscle area. Biochemically, MP decreased glycogenolytic activity, while fatty acid oxidation and oxidative capacity were increased. In conclusion, long-term low-dose MP administration caused a marked impairment in diaphragm function. This is accompanied by changes in diaphragm muscle morphology and enzyme capacity.

Polarised expression of human intestinal N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin) alpha and beta subunits in Madin-Darby canine kidney cells.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.

The nucleoporin CAN/Nup214 binds to both the cytoplasmic and the nucleoplasmic sides of the nuclear pore complex in overexpressing cells.

CAN/Nup214, an essential component of the vertebrate nuclear pore complex (NPC), is required for proper cell cycle progression and nucleocytoplasmic transport. It is a member of the FG-repeat-containing family of nucleoporins and has been localized to the cytoplasmic face of the NPC. Indirect immunofluorescence studies with specific antibodies have shown that moderate overexpression of human CAN in HeLa cells causes an increase in CAN/Nup214 levels at the nuclear envelope. Here, we demonstrate that in such HeLa cells, CAN/Nup214 does not localize exclusively to the cytoplasmic side of the NPC. Cryosections, stained with CAN-specific antibodies and examined by electron microscopy, showed that about one-third of the gold-labeled NPCs were decorated at the cytoplasmic face and the remaining two-thirds at the nucleoplasmic face. These data indicate that both the cytoplasmic fibrils and the nuclear basket of the vertebrate NPC contain specific binding sites for either CAN/Nup214 or for its interacting proteins, Nup88 and hCRM1. Thus, it is conceivable that CAN/Nup214 functions in nucleocytoplasmic transport at both faces of the NPC.

A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention.

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.

Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase.

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the alpha- and beta-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1, 2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 microM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.

Localization and regulation by vitamin D of calcium transport proteins in rabbit cortical collecting system.

The 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced expression of Na+/Ca2+ exchanger, Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase), and calbindin-D28k was investigated in the rabbit distal nephron. Immunocytochemical studies in rabbit kidney sections revealed colocalization of the three Ca2+ transport proteins in the majority of cells in the distal nephron, including connecting tubules and cortical collecting ducts. Subsequently, rabbit connecting and cortical collecting tubule cells were immunodissected and cultured on permeable supports. Immunocytochemical analysis of the cultured cells by confocal microscopy revealed that Na+/Ca2+ exchanger and Ca(2+)-ATPase were present at the basolateral membrane, whereas calbindin-D28k was evenly distributed throughout the cytosol. Concomitant with an increase in Ca2+ transport, 1,25(OH)2D3 increased calbindin-D28k protein and RNA content two- to threefold, as determined by Northern and Western blotting. By contrast, neither Na+/Ca2+ exchanger nor Ca(2+)-ATPase RNA or protein content was noticeably altered. Our findings suggest that 1,25(OH)2D3 stimulation of transcellular Ca2+ transport in primary cultures of rabbit cortical collecting system cells involves an increase in the gene expression of calbindin-D28k but not of Na+/Ca2+ exchanger and Ca(2+)-ATPase.

Effects of different treatment regimens of methylprednisolone on rat diaphragm contractility, immunohistochemistry and biochemistry.

Systemic corticosteroid therapy may affect diaphragm structure and function. We postulated that functional, immunohistochemical and biochemical characteristics of rat diaphragm were less affected by alternate-day methylprednisolone (MP) administration, and more by repeated bursts of MP, in comparison to daily s.c MP. Sixty adult rats were randomized into four groups: saline s.c.; MP continuously (MP-C), 1 mg.kg-1 daily, MP alternate-day therapy (MP-A), 2 mg.kg-1 every other day; or MP in bursts (MP-B), MP 2 mg.kg-1 daily for 2 weeks, saline for 4 weeks, MP 2 mg.kg-1 daily for 2 weeks. The total treatment period was 8 weeks. Contractile properties of isolated diaphragm strips were measured. Antibodies reactive with type I, IIa, IIx and IIb myosin heavy chains were used for immunohistochemical analysis. Biochemical evaluation included markers of fast energy supply, glycogenolytic activity, beta-oxidation capacity and oxidative capacity. The force-frequency curve was depressed in all MP groups. Fibre type I, IIx and IIb cross-sectional area (CSA) decreased in all MP groups. Burst therapy decreased the contribution of type IIb fibres to total diaphragm muscle area. MP-A affected glycogenolytic activity less than MP-C. Burst MP therapy reduced creatine kinase (CK) activity and beta-oxidation capacity compared to MP-C. Oxidative capacity was increased in all MP groups. In conclusion, although the methylprednisolone treatment regimens affected diaphragm muscle morphology and bioenergetic enzyme activities in different ways, force generation decreased in all methylprednisolone treated groups to the same extent.

In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized.

It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this in more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patient's intestinal tissue and from normal controls. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.