Findings consistent with equine proximal suspensory desmitis can be reliably detected using computed tomography and differ between affected horses and controls.

The objective of this retrospective, observational, controlled study was to evaluate bone and soft tissue window CT images of the proximoplantar metatarsus III region in twenty horses with pain localized to the proximal suspensory ligament (PSL) and 20 horses with findings nonrelated to tarsal pain. All horses underwent CT and radiographic examination. Images were reviewed by three independent observers who graded the severity and localization of findings. Bone-related categories as well as soft tissue-related categories were evaluated. For the comparison of imaging findings in horses with and without proximal suspensory desmitis (PSD), mixed linear regression was performed. The intraclass correlation coefficient (ICC) was calculated to assess intraobserver agreement, and kappa statistics were employed to evaluate interobserver agreement. CT examination identified significantly more abnormalities in the diseased group. The scores for osseous exostosis (p = .015) and PSL enlargement (p = .004) were notably higher in PSD horses compared to controls. Intraobserver agreement was overall high (ICC .82-1.0), and interobserver agreement was substantial for the detection of mineralization (kappa = .61) and moderate for sclerosis (kappa = .43), exostosis (kappa = .43), and PSL enlargement (kappa = .48/.51). Measurements in the soft tissue window were significantly smaller than those in the bone window. Findings concurrent with PSD including osseous proliferation and sclerosis as well as soft tissue enlargement, mineralization, and avulsion can be reliably detected using CT. Findings from the current study supported the use of CT for evaluating horses with suspected PSD where high-field MRI is not available.

In vivo corrosion on retrieved hip endoprostheses and in vitro effects of corrosion products on bone mineralization.

In vivo corrosion of modular endoprostheses remains a great concern, as the release of heavy metal ions can impair the implant's service life and the wellbeing of the patient. The detailed corrosion mechanisms that occur in vivo are so far not completely understood. In this context, the effects of implant released cobalt (Co) and chromium (Cr) ions on osteoblast mineralization and gene expression have not been investigated extensively. This comprehensive study aimed at furthering the understanding of in vivo implant corrosion from the clinical signs via prosthesis retrievals and histology of the synovial membranes down to the molecular processes instigated by corrosion products and its effects on bone mineralization. A detailed in vivo failure analysis was performed investigating 22 retrieved hip endoprostheses from different manufacturers and taper material combinations. The aim was to find a correlation of taper damage and especially corrosion to susceptible biomedical alloys and its effect on periprosthetic tissue as well as the clinical implant performance with regard to revision diagnosis and presence of radiolucent lines (RLL). A second part investigated the effects of Co and Cr ions on the in vitro mineralization process of osteoblasts. Cell cultures were exposed to relevant concentrations of CoCl2 and CrCl3 (0 µM, 100 µM, 200 µM) with and without addition of phosphate. Mineralization behavior was analyzed with Alizarin Red assay and Von Kossa staining of calcium depots, alkaline phosphatase activity of osteoblasts and gene expression was analyzed with real time quantitative PCR. The retrieval study provides evidence of in vivo fretting and crevice corrosion on all metallic tapers combined with either ceramic or metal femoral heads. Within the modular taper junctions, selective dissolution of the α phase occurred in wrought TiAl6V4 alloys, and etching of the fine-grained wrought CoCr28Mo6 alloy implants was observed in formed crevices. In addition, significant amounts of wear particles and corrosion products were detected in retrieved synovial membranes. An increased risk for the occurrence of a RLL in the proximal zones was determined for patients with a corroded mixed metal taper. Whereas Co ions have hardly any effects on mineralization, Cr ions cause a significant concentration dependent decrease in mineralization rate of osteoblasts. However, this effect is alleviated by addition of a phosphate source. Our data reveal that Cr ions depleted dissolved phosphates by forming an insoluble complex (CrPO4), which inhibits the phosphate dependent mineralization process. No significant effect of the heavy metal ions on osteoblast activity by means of alkaline phosphate activity as well as on gene expression is determined. This study broadens the understanding of in vivo corrosion of metallic modular implants and its clinically relevant effects on mineralization. Based on these findings, in vivo corrosion of CoCr28Mo6 endoprostheses should be limited to avoid inhibitory effects of Cr3+ on bone mineralization which can contribute to premature implant failure.

Modified toggle pin technique combined with prosthetic capsular reconstruction for surgical stabilization of coxofemoral luxation in a Shetland pony.

OBJECTIVE: To describe open reduction and surgical stabilization of a coxofemoral luxation in a pony using a modified toggle pin technique and prosthetic joint capsule reconstruction without osteotomy of the greater trochanter. ANIMAL: A 2-year-old Shetland pony with a bodyweight of 167 kg. STUDY DESIGN: Case report. METHODS: Radiographic examination confirmed craniodorsal luxation of the left coxofemoral joint. An open reduction with the aid of a pulley system was performed. A toggle pin was inserted through a bone tunnel extending from the level of the femoral shaft through the femoral head and the center of the acetabulum for the pin to be positioned on the medial wall of the acetabulum. FiberWire was subsequently passed through the cranial and caudal aspects of the acetabulum as well as a transverse tunnel in the femoral neck in a figure of 8 to facilitate capsular reconstruction. The pony was placed in a sling for 8 weeks and gradually returned to normal activity over 2 months. RESULTS: Postoperative radiographic examination confirmed the position of the femoral head in the acetabulum with the implants in place. On 2-year follow-up the pony was sound at walk and trot. CONCLUSION: A combined intra- and extra-articular stabilization technique for coxofemoral luxation in a pony resulted in successful long-term reduction and excellent outcome.

Phenotypic Discovery of Triazolo[1,5-c]quinazolines as a First-In-Class Bone Morphogenetic Protein Amplifier Chemotype.

Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Cystatin C serum levels in healthy children are related to age, gender, and pubertal stage.

BACKGROUND: This study aims to establish age- and gender-specific cystatin C (CysC) reference values for healthy infants, children, and adolescents and to relate them to pubertal stage, height, weight, and body mass index (BMI). METHODS: Serum CysC and creatinine levels of 6217 fasting, morning venous blood samples from 2803 healthy participants of the LIFE Child study (age 3 months to 18 years) were analyzed by an immunoassay. Recruitment started in 2011; 1636 participants provided at least one follow-up measurement. Percentiles for CysC were calculated. Age- and gender-related effects of height, weight, BMI, and puberty status were assessed through linear regression models. RESULTS: Over the first 2 years of life, median CysC levels decrease depending on height (ß = - 0.010 mg/l/cm, p < 0.001) and weight (ß = - 0.033 mg/l/kg, p < 0.001) from 1.06 to 0.88 mg/l for males and from 1.04 to 0.87 mg/l for females. Following the second year of age, the levels remain stable for eight years. From 11 to 14 years of age, there is an increase of median CysC levels in males to 0.98 mg/l and a decrease in females to 0.86 mg/l. The change is associated with puberty (ß = 0.105 mg/l/Tanner stage, p < 0.001 in males and ß = - 0.093 mg/l/Tanner stage, p < 0.01 in females) and in males with height (ß = 0.003 mg/l/cm, p < 0.001). CONCLUSIONS: CysC levels depend on age, gender, and height, especially during infancy and puberty. We recommend the use of age- and gender-specific reference values for CysC serum levels for estimating kidney function in clinical practice.

The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu.

SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control.

Choline alkylsulfates--new promising green surfactants.

In this work we show how a new promising green and highly water-soluble surfactant can be designed based on recent progress in the knowledge of counterion-headgroup binding and crystallization behavior. The result is the combination of a most classical surfactant anion, dodecylsulfate (DS), with choline (Ch), a natural green cation. The advantage of the physiological metabolite choline is its bulky structure that prevents ChDS from easy crystallization and thus leads to a considerable lowering of the Krafft point down to 0°C. The counterion-headgroup binding is reflected by the aqueous phase behavior of ChDS. Conductivity, surface tension, and cryo-TEM measurements allow the characterization of the dilute micellar region, while the penetration scan technique enables the establishment of a preliminary aqueous phase diagram. In addition, the influence of different mono- and divalent salts on the solubility of ChDS is investigated. The results are compared to the alkali sulfate and alkylcarboxylate homologs, and reveal that ChDS is less sensitive towards addition of salts than, for instance, choline carboxylates due to an increased counterion-headgroup association. Further, cytotoxicity tests on HeLa and SK-Mel 28 cells are presented and compared to other surfactants, showing that ChDS is no more harmful than its sodium counterpart SDS. Taken together, our findings highlight that the harmless green cation choline is of great potential for the design of new surfactants.

Clinical and functional characterization of a patient carrying a compound heterozygous pericentrin mutation and a heterozygous IGF1 receptor mutation.

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration.

Role of EBAG9 protein in coat protein complex I-dependent glycoprotein maturation and secretion processes in tumor cells.

Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.

Comparative 3'UTR analysis allows identification of regulatory clusters that drive Eph/ephrin expression in cancer cell lines.

Eph receptors are the largest family of receptor tyrosine kinases. Together with their ligands, the ephrins, they fulfill multiple biological functions. Aberrant expression of Ephs/ephrins leading to increased Eph receptor to ephrin ligand ratios is a critical factor in tumorigenesis, indicating that tight regulation of Eph and ephrin expression is essential for normal cell behavior. The 3'-untranslated regions (3'UTRs) of transcripts play an important yet widely underappreciated role in the control of protein expression. Based on the assumption that paralogues of large gene families might exhibit a conserved organization of regulatory elements in their 3'UTRs we applied a novel bioinformatics/molecular biology approach to the 3'UTR sequences of Eph/ephrin transcripts. We identified clusters of motifs consisting of cytoplasmic polyadenylation elements (CPEs), AU-rich elements (AREs) and HuR binding sites. These clusters bind multiple RNA-stabilizing and destabilizing factors, including HuR. Surprisingly, despite its widely accepted role as an mRNA-stabilizing protein, we further show that binding of HuR to these clusters actually destabilizes Eph/ephrin transcripts in tumor cell lines. Consequently, knockdown of HuR greatly modulates expression of multiple Ephs/ephrins at both the mRNA and protein levels. Together our studies suggest that overexpression of HuR as found in many progressive tumors could be causative for disarranged Eph receptor to ephrin ligand ratios leading to a higher degree of tissue invasiveness.

The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex.

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1alpha (EF-1alpha) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1alpha. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.

The navigation-controlled drill in temporal bone surgery: a feasibility study.

BACKGROUND: This study examines the feasibility of a navigation-controlled (NC) drill for surgery on the petrosal bone in an experimental environment. According to the principle of NC, the drill is to be switched off automatically once the borders of the workspace are exceeded during a mastoidectomy. MATERIALS AND METHODS: The registration is based on an optical navigation system with navigation software (MiMed). As surgery engine, the Unidrive-system (Karl Storz GmbH & CO. Kg, Tuttlingen, Germany) was integrated. The definition of the workspace was performed manually in axial computed tomography (CT) slices of the petrosal bone phantom. The mastoidectomy on the model was accomplished in three runs with 10 trial surgeons altogether (5 experienced [exp.] in otologic (ear) surgery, 5 inexperienced [nonexp.]). During each run, the following were logged: the total length of time for the procedure as well as the number and extent of injuries to the risk structures (facial nerve, horizontal semicircular canal, sigmoid sinus). The resultant petrosal bone cavities were measured on the CT. RESULTS: The time for the segmentation of the workspace for the mastoidectomy amounted to 17 minutes. The mean value of the drilling (e.g., milling) performance ranges from 6.61 mm3/s (group 1 [nonexp. + NC]), 9.62 mm3/s (group 2 [exp. w/o NC]), to 10.08 mm3/s (group 3 [exp. + NC]). The relative deviation to the segmented volume amounts to +7.4% (794.3 mm3) for group 1, -39.9% for group 2, and -34% (3,647.0 mm3) for group 3. In the groups with NC guidance of the drill, no damage to a risk structure could be logged. In the group of exp. ear surgeons without NC assistance, one injury to the facial nerve in the petrosal bone phantom occurred. DISCUSSION: The results that follow prove the fundamental feasibility of an NC drill for surgery of the petrosal bone using the example of the simple mastoidectomy in the laboratory test. When using NC, tissue resection is faster, more precise, and has fewer related complications than the same procedure without. The results offer a very promising basis for the introduction of a newly conceived system to the procedure of NC surgery on the petrosal bone. The device configuration used here was originally conceived for NC guidance of a shaver in functional endoscopic sinus surgery. Individual errors will have to be mitigated through the new version of the control unit presently in development.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization.

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

Action and reaction: Chlamydophila pneumoniae proteome alteration in a persistent infection induced by iron deficiency.

Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. Different in vitro models have been established to mimic and characterize chlamydial persistency. For C. pneumoniae and Chlamydia trachomatis, altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most studies including transcriptome and proteome analyses describe persistency induced by IFNgamma treatment. Here, we use iron depletion of the infected cell culture that also leads into persistent infection. We describe differently regulated proteins found by subtractive proteome analysis comparing two early stages of infection with and without addition of the iron chelator deferoxamine-mesylate. While only one bacterial protein was up-regulated during iron deficiency up to 24 h post infection (p.i.), 11 were found to be up-regulated and eight to be down-regulated from 24-48 h p.i. Two down-regulated proteins could be identified by peptide mass fingerprinting as thioredoxin reductase and chromosome partitioning protein (ParB). The latter is involved in chromosome segregation. Thus, using a comparative approach we identified on a proteome level down-regulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae.

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r.

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas.

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.

Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins.

An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.

Generalized crystal-storing histiocytosis associated with monoclonal gammopathy: molecular analysis of a disorder with rapid clinical course and review of the literature.

Crystal-storing histiocytosis (CSH) is a rare event in disorders associated with monoclonal gammopathy. The intracellular crystal formation is almost always accompanied by the expression of kappa light chains. However, the exact mechanism for the storage has not been clarified until now. We report a case of generalized CSH in a 73-year-old man who presented with IgA kappa paraproteinemia and paraproteinuria. The initially observed CSH in the bone marrow biopsy was associated with the clinical and pathomorphologic features of a monoclonal gammopathy of undetermined significance. The progression of disease could not be affected by steroid therapy and the patient died of septic shock 7 months after detection of CSH. At the time of autopsy there was evidence for multiple myeloma and generalized CSH. Two-dimensional gel electrophoresis of liver tissue combined with immunoblotting revealed the massive storage of heavy chains of alpha type and light chains of kappa type, each in a monoclonal pattern. Analysis of the stored kappa light chain by nanoelectrospray-ionization mass spectrometry indicated that it belongs to the variable (kappa)I variability subgroup. We identified some unusual amino acid substitutions including Leu59, usually important for hydrophobic interactions within a protein, at a position where it has never been previously described in plasma cell disorders. In conclusion, we present the first case of CSH with molecular identification of the stored kappa subgroup and detection of unusual amino acid substitutions. Our results suggest that conformational alterations induced by amino acid exchanges represent a crucial pathogenic factor in CSH.