Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

OBJECTIVE: To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation. MATERIALS AND METHODS: Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals. Field-emission scanning electron microscopy was used to assess effects of proteinase K and cathepsin D on internal and superficial crystal structure. RESULTS: Osteopontin was undetectable in COM extracts, but clearly visible in COD. Prothrombin fragment 1 was abundant in COM, but present in COD in lesser amounts than osteopontin. The selectivity was also the same with crystals from ultrafiltered urine that were incubated in centrifuged and filtered urine: prothrombin fragment 1 binding was favoured by low calcium concentration, while osteopontin bound at higher levels. Scanning electron microscopy of COM and COD digested with proteinase K and cathepsin D revealed superficial and internal texture, as wells as surface erosion, in crystals from centrifuged and filtered urine, thus confirming the presence of intracrystalline proteins. Such features were absent from crystals precipitated from ultrafiltered urine. CONCLUSION: Binding of osteopontin and prothrombin fragment 1 to calcium oxalate is dictated primarily by ambient calcium concentration. Each protein may inhibit urolithiasis by inhibiting crystallization of its preferred crystal habit, and by facilitating the intracellular disintegration and dissolution of crystals attached to and internalized by renal epithelial cells.     

Altered exoskeleton composition and vitellogenesis in the crustacean Gammarus sp. collected at polluted sites in the Saguenay Fjord, Quebec, Canada

Gammarus sp. individuals were collected at four intertidal sites subjected to direct sources of pollution (marinas, ferry traffic, and harbors) and at one site with no direct source of pollution. Levels of vitellogenin-like proteins (Vtg), metallothioneins (MT), alkali-labile phosphates (ALPs) in proteins, and lipogenic enzyme activities (i.e., glucose-6-dehydrogenase, isocitrate dehydrogenase, and malate enzyme) were measured in whole soft tissues. In exoskeletons, levels of pH-dependent extractable protein and chitin were determined to assess the possible impacts of pollution on exoskeleton integrity and the molting process. Results show that males were consistently heavier than females regardless of site quality but that the whole-body weight of both sexes was significantly lower at polluted sites. Females displayed either induced or decreased Vtg-like proteins at polluted sites, indicating significant changes in gametogenesis activity. MT levels were not sex dependent and tended to be induced at all impacted sites. ALP levels in acetone-fractionated proteins indicate altered phosphate mobilization at some impacted sites, where females tended to display higher ALP levels. Lipogenic enzyme activities did not vary by sex but were readily increased at impacted sites, suggesting a delay in gonad maturation rates. Exoskeleton protein characteristics revealed that the proportion of chitin in exoskeletons was a lower at most impacted site, suggesting disruption of chitin and pH-dependent protein mobilization. Principal component analysis revealed that gammarids collected at affected sites displayed substantial changes in the proportion of chitin, arthropodin, sclerotin, MTs, and intermediary glucose metabolism (glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase in soft tissues) and thus suffered from disturbed gametogenesis and exoskeleton integrity.     

甲胺磷对母鸡坐骨神经中神经丝蛋白的影响

目的观察甲胺磷诱导母鸡迟发性神经毒性时坐骨神经中神经丝蛋白的变化。方法皮下注射甲胺磷30mg/kg,连续15d。分别在母鸡呈现迟发性神经毒性症状的第2天、第10天和第23天,采用免疫印记法检测坐骨神经沉淀和上清液中神经丝亚单位高分子量神经丝(NF H)、中分子量神经丝(NF M)和低分子量神经丝(NF L)的含量。结果坐骨神经沉淀中,NF H积分密度(IOD)值在呈现症状第2天为145117±17038,比对照组的78875±22569升高了84%,呈现症状第10天和第23天分别为55917±17333和45038±6662,与对照组比较,分别降低了29%和43%;NF M的IOD值在呈现症状第2天时为31493±4625,与对照组的27925±2660比较,差异无统计学意义;呈现症状第10天和第23天分别为19367±2746和6612±1119,明显低于对照组;NF L的IOD值在呈现症状第2天时为39211±3800,明显高于对照组的28749±9319;第10天和第23天分别为27974±3611和21507±2286,与对照组比较差异无统计学意义。3个时间点上清液中NF H的IOD值分别为4709±1739、12337±3205和16745±931,均明显低于对照组的44083±6895;NF M含量明显降低,呈现症状第2天的IOD值为3196±269,比对照组的17243±3232降低了81%,第10天时仅检测到微量,第23天时的IOD值为5206±1292,比对照组降低了70%;呈现症状第2     

Identification of immunodominant autoantigens in rat autoimmune orchitis

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.     

Influence of tissue fixation on the microextraction and identification of amyloid proteins

In surgical pathology, correct immunohistochemical identification of AL amyloidosis poses a particular problem. Immunostaining for lambda- or kappa-light chains is commonly encountered even in non-immunoglobulin-derived amyloidoses, which leads to a false-positive classification as AL amyloidosis. In this respect, microextraction of amyloid proteins from surgical pathology specimens and their subsequent biochemical characterization may prove useful in reaching the correct diagnosis. In this study, we investigated systematically the influence of fixation on the extraction of amyloid proteins from amyloid-containing tissue samples. Tissue samples were obtained from a patient with generalized AA amyloidosis and from a second patient with generalized AL amyloidosis. The samples were stored either unfixed or fixed in phosphate buffered 4% p-formaldehyde, methacarn, or Bouin for 3 days, 1 week, or 1 month. Thereafter, proteins were extracted according to the procedure of Layfield et al, separated by SDS-PAGE and subjected to Western blotting, using antibodies directed against AA amyloid and immunoglobulin-derived lambda-light chain. Following this procedure, a variety of differently sized AA amyloid or lambda-light chain immunoreactive protein bands were found in both patients, which is typical for amyloid proteins. Fixation time did not per se prohibit the extraction of these amyloid proteins from tissue samples, which remained detectable irrespective of fixation time. Although all three fixatives impaired the resolution of some, but not all, individual amyloid proteins, this procedure may help to confirm or reject a diagnosis of AL amyloidosis, because detection of several lambda- or kappa-light chain immunoreactive protein bands in the low-molecular-weight range (<20 kDa) is a common characteristic of their amyloid nature.     

Specific antisera produced by direct immunization with slices of polyacrylamide gel containing small amounts of protein

Rabbits were injected with slices of polyacrylamide gels containing entrapped insect proteins after separation by electrophoresis. Specific antibodies were produced independently of the nature of the gel (with or without sodium dodecyl sulphate) and of the staining technique (amido black or Coomassie Blue). The procedure appears to be a rapid and simple method for production of antibodies specific to proteins separated in minute quantities from a complex mixture.     

Pigeon egg white lysozyme

Lysozyme from pigeon egg white has been purified by ion exchange chromatography and gel filtration. The overall yield of the purification procedure is 65%. The specific activity of the enzyme is 15 000 units/mg. The influence of pH and ionic strength on the lytic activity of the protein, as well as its thermal stability, have been studied. The molecular weight, secondary structure estimation, amino acid composition, NH₂- and COOH-terminal sequence of the protein are also reported. The pigeon enzyme has been classified as a chicken type lysozyme (lysozyme c) according to the obtained results.     

Changes in surface protein structure of bull spermatozoa during epididymal maturation

The surface proteins of bull spermatozoa from caput and cauda epididymidis were labelled by lactoperoxidase-catalyzed radioiodination and solubilized and analysed by SDS-PAG-electrophoresis. The surface protein patterns of caput and cauda epididymal spermatozoa resembled each other but some distinct differences could be found. Caput epididymal spermatozoa revealed a protein peak with molecular weight of 15 000 - 18 000 daltons but this peak was not found on cauda epididymal spermatozoa. On caput epididymal epermatozoa the most intensely labelled protein peak was located between 90 000 and 100 000 daltons but on cauda epididymal spermatozoa the corresponding peak was only weakly labelled and had a molecular weight of 80 000–90 000 daltons. Surface protein with molecular weight of 42 000–47 000 daltons was dominating on cauda epididymal spermatozoa. The surface protein structure of cytoplasmic droplets did not drastically differ from that of epididymal spermatozoa.