Oxidative and nitrosative stress induces peroxiredoxins in pancreatic beta cells.

AIMS/HYPOTHESIS: Insulin-producing beta cells are destroyed by oxidative and nitrosative stress during the pathogenesis of Type I (insulin-dependent) diabetes mellitus. These cells are more sensitive than others due to their deficiency of well known antioxidant enzymes like superoxide dismutase, glutathione peroxidase and catalase. However the peroxiredoxins discovered in the past decade form a large family of highly conserved thioredoxin-dependent peroxide reductases, which are present in most tissues. We investigated whether peroxiredoxins I and II are present in pancreatic beta cells and if they are inducible by oxidative and nitrosative stress. METHODS: To detect these enzymes in insulin-producing beta cells we used semiquantitative RT-PCR, western blots and immunohistochemistry. The expression of peroxiredoxins I and II was analysed after treatment with cytokines, hydrogen peroxide, alloxan or streptozotocin in the rat insulinoma cells INS-1 using RT-PCR and western blots. RESULTS: We show that peroxiredoxins I and II are present in the cytoplasm of pancreatic islet cells as well as in insulinoma cell lines beta TC6-F7 and INS-1. Peroxiredoxins I and II were up-regulated by all stress agents used. CONCLUSION/INTERPRETATION: Beta cells, undersupplied with well characterized antioxidant enzymes, possess an additional antioxidant system which is inducible by oxidative as well as nitrosative stress.

Developmental expression of sodium entry pathways in rat nephron.

During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.

Structural and molecular dissection of the juxtaglomerular apparatus: new aspects for the role of nitric oxide.

The juxtaglomerular apparatus (JGA) is composed of the macula densa (MD), the extraglomerular mesangium, and the juxtaglomerular arterioles. The JGA functions to adapt glomerular filtration rate (GFR) to distal tubular [NaCl] and to adjust the synthesis and release of renin. The type 1 isoform of nitric oxide synthase (NOS1) is present in MD cells, and release of NO toward the glomerular vasculature is thought to modulate signaling at the JGA. Chronic alterations in GFR and/or tubular [NaCl] are paralleled by adjustments of NOS1. Molecular characterization of NOS1 mRNA reveals several renal variants suggesting cell type-specific regulation at the level of transcription and translation.

Nitric oxide synthase 1 mRNA: tissue-specific variants from rat with alternative first exons.

We cloned a novel alternative first exon for nitric oxide synthase 1 (NOS1) that is specific for kidney and two novel alternative second exons which can be inserted between the kidney-specific first exon and the exon currently numbered exon 2. The novel exons were localized within 17 kb upstream of exon 2, and their flanking regions and the boundaries of exon 2 were sequenced. NOS1 mRNAs starting with four additional alternative first exons were characterized with respect to tissue distribution and alternative splicing. Altogether, at least 11 different splice variants were found. Those present in kidney were mainly lacking exon 2.

In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle.

Skeletal muscle provides a major source of the signaling molecule nitric oxide (NO) however in situ identification of NO-synthase (NOS) mRNA has not been verified. We have used NOS-I (neuronal NOS) probes prepared from plasmid DNA by reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA transcripts in skeletal muscle cells and myofibers of rat and mouse. Mouse C2C12 myoblasts and myotubes reveal strong cytosolic in situ hybridization (ISH) signals in vitro. In adult animals, ISH signals are detectable in striated myofibers at subsarcolemmal and perinuclear regions whilst the myofibrillar compartment is devoid of signals. Expression of NOS-I mRNA in fusion-competent myoblasts suggests that the NOS/NO system is of relevance to myogenic differentiation. Compartmentalization of NOS-I mRNA may reflect spatiofunctional actions between NOS message and protein and the putative subcellular NO targets.

Characterization of the Han:SPRD rat model for hereditary polycystic kidney disease.

The Han:SPRD rat model for inherited polycystic kidney disease (PKD) was characterized (clinical parameters, morphology, immunohistochemistry and in situ hybridization). Homozygous animals died of uremia after three to four weeks with severe cystic transformation of virtually all nephrons and collecting ducts (serum urea: 616 +/- 195 mg/dl; kidney-to-body weight ratio: > 20%). In heterozygotes, slow progression of the disease led to death between the 12th and 21st month (median: 17 months; serum urea levels above 200 mg/dl). Kidney enlargement was moderate, and cysts were restricted to the cortex and outer medulla. Immunohistochemical markers showed that approximately 75% of the cysts were derived from the proximal tubule. Cystic transformation started in the proximal tubule with a sharp onset of basement membrane alteration and a loss of epithelial differentiation restricted to small focal areas. In these areas, alpha 1(IV) collagen and laminin B1 mRNA were enhanced as revealed by isotopic and non-isotopic in situ hybridization. Fibroblasts underlying the affected tubular portions were involved in matrix overexpression resulting in subepithelial accumulation of immunoreactive collagen IV and laminin. In later stages of cystic transformation distal nephron segments were affected as well. A reversal in epithelial polarity as judged from Na,K-ATPase-immunoreactivity was not observed. Renal immunoreactive renin-status was significantly decreased. Hematocrit was lowered in heterozygotes (40.4 +/- 5.8 vol% compared to 46.7 +/- 1.99 vol% in controls; P < 0.05) and total renal EPO mRNA was reduced to 36 +/- 14% of the mean value of control animals, whereas serum EPO levels were not significantly altered. We conclude that the Han:SPRD rat is a useful model for the study of human ADPKD since both diseases are similar in several aspects. The model is particularly suitable for the study of epithelial-mesenchymal interactions at the beginning of tubular cystic transformation.

Focal overexpression of collagen IV characterizes the initiation of epithelial changes in polycystic kidney disease.

Changes of extracellular matrix are involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The relationship between epithelial changes and extracellular matrix production was studied in a new rat model (Han:SPRD/cy+) at an early phase of cystogenesis. Messenger RNA expression of the alpha 1(IV)-chain of collagen type IV, the main structural component of basement membrane, was localized by in situ hybridization. The presence of collagen IV-protein was shown by immunohistochemistry. At an initial stage, cysts were lined with normal-appearing epithelium except for focal zones of less differentiated cells exhibiting strong collagen alpha 1(IV) mRNA expression and a thickened basement membrane. In these zones, an increase in cell number (2.39-fold) per unit epithelial area indicated hyperplastic growth. Conspicuously, these zones were found at 'bottleneck'-like transitions from normal-size tubules to cystic expansions. Intermediate stages of cysts showed more prominent extracellular matrix deposits and an overall maximally enhanced collagen IV mRNA expression, whereas terminal stages were lined with a flat, simplified epithelium and exhibited moderate collagen IV expression. We suggest that focally enhanced expression of collagen IV in the tubular epithelium and surrounding interstitium of Han:SPRD/cy+ rat kidney is initially involved in cyst development.

Expression of the laminin-A chain is down-regulated by a non-canonical polyadenylation signal.

It is well accepted that 3' untranslated regions (UTR) are an essential part of mRNA. However, little is known in detail about the contribution of different regions of 3' UTR on synthesis, stability and translatability of their mRNA. In addition to the highly conserved hexanucleotide AAUAAA, some consensus sequences for 3'-end processing and polyadenylation have been characterized, but most of this work has been done with viral mRNA or beta-globin mRNA. We have studied the influence of the 3' UTR of the mRNA for the three chains A, B1, B2 of laminin on the expression of a reporter gene (galK). Laminin is a large glycoprotein of basement membranes and all three polypeptide chains are needed in equal amounts for a functional molecule. The three 3' UTR of the laminin mRNA differ widely with respect to length, number of polyadenylation signals and other consensus sequences. Nevertheless, all three 3' UTR reduce the expression of the reporter gene at least three-fold, when the corresponding cDNA sequences are inserted downstream of the reporter gene instead of the 3' UTR of simian virus 40 early genes. The 3' UTR of laminin-A mRNA contains the non-canonical polyadenylation signal AUUAAA which seems to be responsible for the limiting amounts of laminin-A mRNA and protein compared to those for laminin B1 and B2 [Speth and Oberbäumer (1993) Exp. Cell Res. 204, 302-310]. Mutation of the laminin-A polyadenylation signal to the canonical form AAUAAA increases expression by a factor of 2.5.

Expression of basement membrane proteins: evidence for complex post-transcriptional control mechanisms.

Differentiated murine teratocarcinoma cell lines have been widely used as sources for the basement membrane proteins laminin and collagen IV. In order to understand the control of their expression, we have measured the transcription rates of the corresponding genes in nuclear run-on assays. The ratios of transcripts obtained from the five different genes of interest (for laminins A, B1, and B2 and alpha 1(IV), alpha 2(IV)) are rather different from the ratios of the corresponding mRNAs, which are again different from the protein levels needed. The gene for alpha 2(IV) is transcribed at a higher rate than the one for alpha 1(IV) and, similarly, the gene for laminin A is transcribed at a higher rate than the other two laminin genes, respectively. However, the alpha 2(IV) and laminin A mRNA levels are lower than those for the other chains of the same molecule. The alpha 1(IV) mRNA is 3- to 15-fold more abundant than the alpha 2(IV) mRNA, depending on the cell line. At the protein level, the A chain seems to be limiting for the assembly of laminin, in accordance with its low mRNA level. The two collagen chains have variable pool sizes, but the triple helical molecules always seem to be composed of two alpha 1(IV) and one alpha 2(IV) chains. These results point to extensive control mechanisms at various stages of posttranscriptional events, some of which we could identify.

Evaluation of mRNA steady-state and protein levels for basement membrane proteins in cultured murine cells.

We have determined the mRNA steady-state levels for the six constituent polypeptide chains of the basement membrane proteins collagen IV, laminin and nidogen in murine cell lines derived from a teratocarcinoma, and in some other cell lines of different origin in stationary cultures and during different growth phases. The mRNA and protein levels change in response to growth phase. The amounts of the mRNAs for the single chains do not agree with the ratios needed for the different peptide chains of collagen IV and laminin. While the mRNA and protein levels for laminin are in a similar range for the teratocarcinoma-derived cell lines, the mRNA and protein levels vary by at least a factor of 10 for collagen IV. These results point to complex posttranscriptional regulatory mechanisms for the biosynthesis of basement membrane proteins.

Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene.

Several cDNA clones were isolated from cDNA libraries constructed with mRNA longer than 28S RNA from the murine cell line PYS-2/12. The plasmids have inserts containing 1-1.2 kb of the ribosomal 5' external transcribed spacer followed by nearly 700 nt of sequence for 18S rRNA and ending with a B2 element (retroposon). The cloned sequence differed in a few positions from published ribosomal sequences. The 3' adjacent genomic sequence was obtained by polymerase chain reaction (PCR) and showed that the B2 element has a poly(A) tail of about 50 nt and is surrounded by perfect direct repeats of 15 nt. Analysis of genomic DNA from several murine cell lines revealed that PYS cells contain at least one copy of 18S RNA with the B2 element which is not present in the genome of other murine cell lines derived from the same teratocarcinoma. Similarly, rRNA transcripts containing the B2 element were only detected in PYS cells. According to the publication dates of the different cell lines, the B2 element must have been integrated into an rRNA transcription unit during the years 1970 through 1974 thus proving that retroposons (SINEs) can still be inserted into the genome in our times.

DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor.

Oligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations.

Detection of RNA on northern blots by negative staining with aurintricarboxylic acid.

Aurintricarboxylic acid (ATA) is a well-known inhibitor of RNA and DNA modifying enzymes and was suggested as a potent RNase inhibitor for preparation of RNA (Hallick et al., 1977, Nucleic Acids Res. 4, 3055-3064). We show that ATA is a very useful stain for detecting RNA on Northern blots and slot blots although it did not fully protect purified RNA in concentrated solution against RNase A.

Structural study of long arm fragments of laminin. Evidence for repetitive C-terminal sequences in the A-chain, not present in the B-chains.

The outer segments of the long arm of laminin have recently been shown to mediate attachment of many cell types and to stimulate neurite outgrowth. For a structural characterization of this part of the molecule we prepared, by limited elastase digestion of laminin, fragments E3 and E8, previously identified as a globular heparin-binding domain and as a 35-nm-long rod with a terminal globule, respectively. Fragment E3 is a domain adjacent to fragment E8. Both structures together comprise the complete terminal half of the long arm. Our data confirm current models, which predict that the C-terminal segments from all three chains contribute to its structure. The B chains terminate at the end of the rod like domain, while the large terminal globule is formed by A-chain structures only. In addition to fragment E3, two new fragments T1 and T2 obtained by tryptic cleavage of fragment E8 were characterized as substructures of the globular domain. Screening of a mouse cDNA library with synthetic oligonucleotides allowed isolation of an 1.8-kb cDNA clone encoding 547 C-terminal amino acids of the A chain and some 196 nucleotides of the 3'-untranslated region including a single polyadenylation site. The clone contained portions of domain T2 and the complete heparin binding domain E3 which was thus identified as the most C-terminal domain of the A chain. Sequence alignment indicated that the terminal globule is formed by homologous repeats of some 140 residues having no counterpart in the B chains.

The N terminus of laminin A chain is homologous to the B chains.

A major proteolytic fragment (E1/E1-4) of the basement membrane protein laminin, comprising the three short arms with some terminal globules missing, was isolated by elastase digestion, and partial protein sequence data were determined for several tryptic peptides. Sequences which corresponded to A-chain structures were used to synthesize oligonucleotides for the construction and screening of a primer-extended cDNA library from mouse PYS-2 cells. A clone of 1.1 kb was obtained and shown by sequencing to correspond to the 5' end of the 10-kb mRNA of the A chain of laminin. The clone contains 77 nucleotides of 5' untranslated sequence and a region coding for 334 amino acids, including a presumptive signal peptide of 24 amino acids. The sequence is 30% homologous to the corresponding N-terminal part of the B1 chain of laminin, suggesting the same structure for both domains. The data present further evidence for a recent structural model which postulates that each of the three laminin polypeptide chains forms a distinct short arm.

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain.

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.

Completion of the amino acid sequence of the alpha 1 chain of human basement membrane collagen (type IV) reveals 21 non-triplet interruptions located within the collagenous domain.

The cDNA and protein sequences of the N-terminal half of human basement membrane collagen (type IV) have been determined. Overlapping cDNA clones were constructed by repeated primer extension with synthetic oligonucleotides. They cover 2953 bp, beginning at the 5' end of the corresponding mRNA. At the protein level, the sequence of the cyanogen bromide peptide CB6 adjacent to the 7S domain has been additionally elucidated. The data presented here complete the protein sequence and nearly the entire cDNA sequence of the human alpha 1(IV) chain. The amino-terminal half of the alpha 1(IV) chain contains 8 cysteine residues involved in intramolecular and intermolecular cross-links. The entire triple-helical domain of alpha 1(IV) is interrupted by 21 non-triplet regions.

cDNA and protein sequence of the NC1 domain of the alpha 2-chain of collagen IV and its comparison with alpha 1(IV).

We present the complete cDNA and derived amino acid sequence of the non-collagenous domain NC1 of alpha 2(IV). Comparison with the corresponding NC1 domain of alpha 1(IV) reveals a high degree of homology at the protein level, in contrast to the barely homologous triple-helical sequences of both chains.

Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain.

The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen.