Disturbance of inorganic phosphate metabolism in diabetes mellitus: its relevance to the pathogenesis of diabetic retinopathy.

Early in the progression of diabetes, a paradoxical metabolic imbalance in inorganic phosphate (Pi) occurs that may lead to reduced high energy phosphate and tissue hypoxia. These changes take place in the cells and tissues in which the entry of glucose is not controlled by insulin, particularly in poorly regulated diabetes patients in whom long-term vascular complications are more likely. Various conditions are involved in this disturbance in Pi. First, the homeostatic function of the kidneys is suboptimal in diabetes, because elevated blood glucose concentrations depolarize the brush border membrane for Pi reabsorption and lead to lack of intracellular phosphate and hyperphosphaturia. Second, during hyperglycemic-hyperinsulinemic intervals, high amounts of glucose enter muscle and fat tissues, which are insulin sensitive. Intracellular glucose is metabolized by phosphorylation, which leads to a reduction in plasma Pi, and subsequent deleterious effects on glucose metabolism in insulin insensitive tissues. Hypophosphatemia is closely related to a decrease in adenosine triphosphate (ATP) in the aging process and in uremia. Any interruption of optimal ATP production might lead to cell injury and possible cell death, and evidence will be provided herein that such cell death does occur in diabetic retinopathy. Based on this information, the mechanism of capillary microaneurysms formation in diabetic retinopathy and the pathogenesis of diabetic retinopathy must be reevaluated.

Individual variations in protective effects of experimentally formed salivary pellicles.

Salivary proteins protect teeth against acid-induced softening and demineralization by forming a pellicle. However, little is known about individual, gender and ethnic variations in this effect. Therefore, we aimed to determine differences in protective effects of experimentally formed pellicles from 10 healthy young Scandinavians (3 women and 7 men) and 10 healthy young non-Scandinavians (4 women and 6 men) including Arabic, Persian, Pakistan, Indian, and Chinese subjects. Bovine enamel blocks, which were precoated with parotid and submandibular salivary proteins for 12 h, were exposed to an acidic solution with surface microhardness (SMH) determinations before and after. No change in SMH equalled 100% protection, whereas SMH corresponding to no protein coating equalled 0%. The results showed that experimentally formed pellicles from non-Scandinavians protected enamel better than pellicles from Scandinavians (p < 0.001). Within groups protective effects of pellicles formed from parotid and submandibular saliva were equal and subjects with high protection from parotid saliva pellicles also had high protection from submandibular saliva pellicles (r = 0.78; p < 0.001). Within groups considerable differences were obtained among individuals ranging from 25 to 51% protection. However, SDS-PAGE and HPLC did not reveal any systematic relation between saliva protein composition and protective effects, although slightly more of the SN-isoform of S-type cystatin was found in pooled parotid saliva from those non-Scandinavian subjects showing highest protection. We conclude that individual variations in experimental pellicle protection against erosive challenges exist and that such variations appear not to be due to differences in a single protein component.

Potential circulating biomarkers for abdominal aortic aneurysm expansion and rupture--a systematic review.

BACKGROUND: The maximal diameter of abdominal aortic aneurysms (AAAs) is the dominating indication for repair. However half of the AAAs repaired would never have ruptured if left unrepaired, although small AAAs occasionally rupture. Earlier surgery may be associated with a lower mortality. More precise indicators for surgery are warranted. This systematic review identifies potential systemic biomarkers for AAA rupture or expansion. METHODS: MEDLINE/PubMed and EMBASE (from 1985 trough May 2007) were searched with the medical subject heading abdominal aortic aneurysm and keywords "size", "progression" or "growth" or "expansion rate" or "rupture" on the basis of MESH tree and as a text search restricted to English, German, French and Italian. In addition, reference lists were studied and manual searches performed. Observational studies investigating the association of circulating biomarkers with AAA rupture, expansion or size were selected. DATA EXTRACTION: Two reviewers (SU and GU) independently extracted the following data: year of publication, study characteristics, duration of follow-up, circulating biomarker, AAA expansion rate or size or rupture. RESULTS: 699 papers were identified. After exclusion of thoracic aneurysms and cardiac studies (n=118), surgical or medical treatment studies (n=179), case reports and animal studies (n=87), as well as reviews or letters (n=66), 249 articles were selected. Also excluded were 230 papers that did not report AAA size, expansion rate or rupture. 39 papers were included. Several potential biomarkers were identified. The strongest association with AAA was obtained with serum elastin peptides (SEP) and plasmin-antiplasmin (PAP) complexes. Matrix-degrading metalloproteinase 9 (MMP9) and interferon-gamma (IFN-gamma) could have clinical potential while many putative biomarkers showed poor association. CONCLUSIONS: Several circulating agents in peripheral blood may predict AAA size, expansion rate or rupture. Few of them have clinical potential for future use. Confirmative studies and development of multivariate models are needed, together with continuing search for new biomarkers using the discovery based sciences within proteomics and/or genomics.

Characterization of proteinuria and tubular protein uptake in a new model of oral L-lysine administration in rats.

Intravenous infusion of basic amino acids is used experimentally and pharmacologically to prevent renal proximal tubular uptake of filtered proteins. Intravenously injected L-lysine is rapidly cleared from plasma and the effect on tubular protein reabsorption is transient. To obtain a more sustained effect, we developed a model of oral L-lysine administration and characterized this model by analyzing urinary protein excretion and proximal tubule uptake of filtered proteins. Rats placed in metabolic cages were treated with 20 mmol/kg/6 h of L-lysine, glycine, or water. Urines were analyzed for proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and radioimmunoassay. Proximal tubule uptake of proteins and expression of apical membrane receptors were investigated by immunocytochemistry. In vitro uptake and receptor expression were studied using a yolk sac cell line. L-lysine administration produced increased urinary excretion of a large number of proteins while the effect on tubular accumulation of selected proteins was variable. L-lysine treatment induced changes in the localization of two receptors responsible for tubular endocytosis of filtered proteins. In conclusion, oral L-lysine treatment induced proteinuria, in particular albuminuria, as efficiently as previous reports on intravenous infusion. The effect on tubular protein accumulation was variable suggesting differential effects on tubular reabsorption and degradation of filtered proteins. Changes in tubular protein handling were accompanied by changes in the localization of the endocytic receptors, megalin, and cubilin. In vitro experiments supported the in vivo observations. The findings suggest that L-lysine may affect receptor trafficking in addition to possible effects on the direct binding of ligands to the receptors.

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

Immunoblotting analysis of abdominal aortic aneurysms using antibodies against Chlamydia pneumoniae recombinant MOMP.

OBJECTIVES: antibodies against Chlamydia pneumoniae have been associated with atherosclerosis and with expansion of abdominal aortic aneurysms (AAA). C. pneumoniae has been demonstrated in coronary arteries, AAA and the carotid arteries by use of polymerase chain reactions (PCR), immunohistochemical procedures and electron microscopy. However, the correlation between demonstrating C. pneumoniae DNA or antigen in tissue from plaque material or aneurysms and the antibody titres in serum is controversial. The specificity of immunohistochemical procedures is unknown. The aim of this study was to assess the possibility of potential non-specific findings for methods based on immunostaining. MATERIALS AND METHODS: twenty patients undergoing infrarenal AAA repair were studied. Full AAA thickness tissue was collected from the anterior wall of the aneurysm. Analysis was performed using polyacrylamide gelelectrophoresis, immunoblotting and mass spectrometric protein identification. RESULTS: C. pneumoniae antigen was not demonstrated in any of the AAA samples, whereas a major cross-reacting protein was present in all AAA samples. The protein was identified as the human haemoglobin beta chain. CONCLUSION: we were not able to find C. pneumoniae antigens reacting with an anti C. pneumoniae major outer membrane protein (MOMP). Direct detection of C. pneumoniae by immunohistostaining procedures should be interpreted with caution due to potential crossreaction with non chlamydial proteins.

Efficient eukaryotic expression system for authentic human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) is the main carrier for androgens and oestrogens in humans. It mediates the transport of steroid hormones in the circulation and testicular fluid, and regulates their bioavailability to steroid-responsive tissues. In addition, the protein interacts with membrane receptors expressed in target tissues. Binding to the receptors is suspected to facilitate the uptake of steroid hormones and/or elicit cellular signal transduction. The identity of the SHBG receptor has not yet been resolved, in part due to a lack of sufficient quantities of authentic SHBG for receptor purification and molecular characterization. We have successfully addressed this problem by establishing an episomal expression system in human embryonic kidney cells that produces 5 mg of fully active human SHBG per litre. The recombinant protein resembles native SHBG in terms of structure, glycosylation pattern and steroid-binding activity. Moreover, the protein interacts with plasma membranes in steroid target tissues, an activity not observed with SHBG from other recombinant expression systems. Thus our studies have removed an important obstacle to the further elucidation of the role SHBG plays in steroid hormone action.

Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization.

alpha-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate alpha-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca(2+)-regulated processes. A microdialysis technique was applied showing that alpha-synuclein binds Ca(2+) with an IC(50) of about 2-300 microm and in a reaction uninhibited by a 50-fold excess of Mg(2+). The Ca(2+)-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue beta-synuclein, as shown by Ca(2+) binding to truncated recombinant and synthetic alpha-synuclein peptides. Ca(2+) binding affects the functional properties of alpha-synuclein. First, the ligand binding of (125)I-labeled bovine microtubule-associated protein 1A is stimulated by Ca(2+) ions in the 1-500 microm range and is dependent on an intact Ca(2+) binding site in alpha-synuclein. Second, the Ca(2+) binding stimulates the proportion of (125)I-alpha-synuclein-containing oligomers. This suggests that Ca(2+) ions may both participate in normal alpha-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies.

Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals.

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.

Immunolocalization of electroneutral Na-HCO(3)(-) cotransporter in rat kidney.

An electroneutral Na-HCO(3)(-) cotransporter (NBC(N)1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBC(N)1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBC(N)1. The affinity-purified antibody specifically recognized one band, approximately 180 kDa, in rat kidney membranes. Peptide-N-glycosidase F deglycosylation reduced the band to approximately 140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBC(N)1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBC(N)1 immunolabeling. Immunoelectron microscopy demonstrated that NBC(N)1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2, 7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO(3)(-)-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBC(N)1. The localization of NBC(N)1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBC(N)1 may be important for electroneutral basolateral HCO(3)(-) transport in these cells.

Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family.

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na(+) and K(+) concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, alpha and beta. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na, K-ATPase alpha-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the alpha-subunit of pig kidney Na, K-ATPase, indicating specific association with different alpha-isoforms. Finally, PLMS and the alpha-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C(12)E(8) resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.

Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor.

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.

Calumenin interacts with serum amyloid P component.

We recently reported the identification of human calumenin, a novel Ca(2+) binding, transformation-sensitive and secreted protein [Vorum et al. (1998) Biochim. Biophys. Acta 1386, 121-131; Vorum et al. (1999) Exp. Cell Res. 248, 473-481] belonging to the family of multiple EF-hand proteins of the secretory pathway that include reticulocalbin, ERC-55, Cab45 and crocalbin. In order to further investigate the extracellular functions of calumenin we immobilized the recombinant protein to a column. After application of a placental tissue extract we were able to elute one protein that interacts with calumenin in the presence of Ca(2+). Amino acid sequencing identified this protein as serum amyloid P component (SAP). Furthermore, we verified and characterized the calumenin-SAP interaction by the surface plasmon resonance technique. The findings indicate that calumenin may participate in the immunological defense system and could be involved in the pathological process of amyloidosis that leads to formation of amyloid deposits seen in different types of tissues.

The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells.

The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca(2+)-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation.

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

hnRNPs H, H' and F behave differently with respect to posttranslational cleavage and subcellular localization.

hnRNPs H, H' and F belong to a subfamily of the hnRNPs sharing a high degree of sequence identity. Eukaryotic expression and specific C-terminal antibodies were used to demonstrate great variation in the intracellular fate of the proteins. hnRNPs H and H' become posttranslational cleaved into C-terminal 35 kDa proteins (H(C), H'(C)) and possibly into N-terminal 22 kDa proteins. No detectable cleavage was observed for hnRNP F. hnRNP H/H' is almost exclusively localized to the nucleus of many cell types while hnRNP F varies from a predominant nuclear localization in some cells to a predominant cytoplasmic localization in other cells. The different fates may reflect differences in functional roles that so far only have included nuclear functions. The presence of significant quantities of hnRNP F in the cytoplasm of many cells indicates that it also may have a functional role here.

Molecular basis of indomethacin-human serum albumin interaction.

Studies on the strength and extent of binding of the non-steroidal anti-inflammatory drug indomethacin to human serum albumin (HSA) have provided conflicting results. In the present work, the serum-binding of indomethacin was studied in 55 mM sodium phosphate buffer (pH 7.0) at 28 degrees C, by using a fluorescence quench titration technique. The interaction of indomethacin with human serum albumin has been studied as a function of temperature, ionic strength and pH. The results suggest that electrostatic interaction plays a major role in the binding. The possible role of lysine residues in this interaction was studied by modifying exposed and buried lysine residues of HSA with potassium cyanate and studying indomethacin binding with the modified HSA. The data suggest that the interaction takes place via a salt bridge formation between the carboxylate group of indomethacin and a buried lysine residue of HSA. A technique involving fluorescence enhancement of bilirubin upon its interaction with HSA was used to study its displacement by indomethacin. The displacement, although apparently competitive in nature, was not strong suggesting that the primary sites of interaction of bilirubin and indomethacin are different.