Anchoring on Hyperglycemia and Sepsis in the Presence of an Unforeseen Thyroid Storm.

Thyroid storm (TS) is a relatively rare but life-threatening complication of an overactive thyroid that can manifest in a myriad of ways due to its multisystem involvement. Due to its relatively high mortality rate, it is essential that TS is recognized and treated promptly. TS can occur due to trauma, drugs, and sepsis. Identifying TS as a diagnosis is challenging to pinpoint due to its similar presentation to more common pathologies like sepsis and diabetic ketoacidosis (DKA). Here, we present a case of a 31-year-old African-American woman with type 2 diabetes mellitus following sepsis secondary to Escherichia coli pyelonephritis and DKA. Despite standard sepsis treatment, which included appropriate intravenous fluids and antibiotics, the patient did not improve. Further workup, utilizing the Burch-Wartofsky score, helped identify TS as the underlying cause of the patient's hospitalization, despite no history of underlying thyroid disease. The inclusion of thyroid pathology as part of the differential diagnosis and workup of a patient with a sepsis-like presentation to avoid anchoring bias warrants further investigation.

Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase.

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Carbonic anhydrase I, II, and VI, blood plasma, erythrocyte and saliva zinc and copper increase after repetitive transcranial magnetic stimulation.

INTRODUCTION: Repetitive transcranial magnetic stimulation (rTMS) has been used to treat symptoms from many disorders; biochemical changes occurred with this treatment. Preliminary studies with rTMS in patients with taste and smell dysfunction improved sensory function and increased salivary carbonic anhydrase (CA) VI and erythrocyte CA I, II. To obtain more information about these changes after rTMS, we measured changes in several CA enzymes, proteins, and trace metals in their blood plasma, erythrocytes, and saliva. METHODS: Ninety-three patients with taste and smell dysfunction were studied before and after rTMS in an open clinical trial. Before and after rTMS, we measured erythrocyte CA I, II and salivary CA VI, zinc and copper in parotid saliva, blood plasma, and erythrocytes, and appearance of novel salivary proteins by using mass spectrometry. RESULTS: After rTMS, CA I, II and CA VI activity and zinc and copper in saliva, plasma, and erythrocytes increased with significant sensory benefit. Novel salivary proteins were induced at an m/z value of 21.5K with a repetitive pattern at intervals of 5K m/z. CONCLUSIONS: rTMS induced biochemical changes in specific enzymatic activities, trace metal concentrations, and induction of novel salivary proteins, with sensory improvement in patients with taste and smell dysfunction. Because patients with several neurologic disorders exhibit taste and smell dysfunction, including Parkinson disease, Alzheimer disease, and multiple sclerosis, and because rTMS improved their clinical symptoms, the biochemical changes we observed may be relevant not only in our patients with taste and smell dysfunction but also in patients with neurologic disorders with these sensory abnormalities.

Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

The Akt C-terminal modulator protein is an acyl-CoA thioesterase of the Hotdog-Fold family.

Herein, we report on an in vitro kinetic activity analysis that demonstrates that the protein known as the Akt C-terminal modulator protein is a broad-range, high-activity acyl-CoA thioesterase. In vitro tests of possible activity regulation by product inhibition or by Akt1 binding gave negative results. Truncation mutants confined the thioesterase activity to the C-terminal domain, consistent with our threading model. The N-terminal domain of unknown fold and function was found to contribute to solubility.

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

Reexamination of the cysteine residues in glucocerebrosidase.

Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure.

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma.

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.

Detection of human MCP-4/CCL13 isoforms by SELDI immunoaffinity capture.

Monocyte Chemoattractant Proteins 4 (MCP-4/CCL13) is a member of a distinct, structurally-related subclass of CC chemokines mainly involved in recruitment of eosinphils to inflammatory sites. Recent evidence demonstrates that serum level of this protein strongly increases following high dose IL-2 immunotherapy. The physiological form of human MCP-4/CCL13 has yet to be purified. Therefore, the primary structure of the biologically relevant (mature) form has not been established. By using SELDI immunoaffinity capture technology we describe two mature isoforms both present in serum before and after high-dose IL-2 immunotherapy.

Diverse range of small peptides associated with high-density lipoprotein.

High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By HPLC and mass spectrometry, more than 100 peptide components were found in the size range from 1000 to 5000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, alpha1-proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation.

Kinetic analysis of Pseudomonas aeruginosa arginine deiminase mutants and alternate substrates provides insight into structural determinants of function.

L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the l-arginine C(alpha)NH(3)(+)(COO(-)) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N(omega),N(omega)-dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF.

Inflammatory protein profile during systemic high dose interleukin-2 administration.

Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers.

Effect of nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin.

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.

Nitration and inactivation of IDO by peroxynitrite.

IDO induction can deplete L-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-gamma in human macrophages, dendritic cells, and bone marrow cells. L-tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-gamma-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids.

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.

Gastrointestinal beta2microglobulin amyloidosis in hemodialysis patients: biochemical analysis of amyloid proteins in small formalin-fixed paraffin-embedded tissue specimens.

We present here a first report on the biochemical analysis of intestinal amyloid deposits found in two cases of hemodialysis-related amyloidosis. A new microtechnique was applied for extraction and immunochemical/chemical characterization of amyloid proteins in small amounts of fixed tissue, thus allowing precise identification of beta2microglobulin amyloid (Abeta2M) in both cases studied. The molecular mass of the identified amyloid beta2M was close to that of intact beta2M (12 kDa), with no evidence of the products of proteolytic fragmentation of these molecules. The isoelectrofocusing of the purified Abeta2M demonstrated a shift to more acidic pI as compared to the normal beta2M analyzed under the same experimental conditions. The obtained data suggest that the intestinal amyloid deposits in dialysis-related amyloidosis contain disease-specific beta2M isoforms, which could play a role in the pathogenesis of amyloid disease. The new methodology used might be useful in obtaining precise diagnosis of amyloidosis that is necessary for appropriate therapy, and also provide new important information on the chemical structure of amyloid proteins.

Co-deposition of amyloidogenic immunoglobulin light and heavy chains in localized pulmonary amyloidosis.

Localized pulmonary amyloidosis is a rare condition whose pathogenesis is insufficiently understood. In the present study, we report a case of localized pulmonary amyloidosis associated with lung-restricted lymphoplasmacytoid lymphoma, monoclonal for immunoglobulin (Ig) G lambda (lambda). Biochemical microtechniques have been applied for extraction, purification, and characterization of amyloid proteins. Surprisingly, chemical analysis of these proteins revealed a not-previously-described case of combined deposits containing Ig fragments of gamma heavy chain (variable domain) and lambda light chain (constant domain). In view of the absence of circulating monoclonal Ig, this case supports the hypothesis that localized amyloid is formed by local plasmacytoid cells.

A newly discovered human alpha-globin gene.

A previously undefined transcript with significant homology to the pseudo-alpha2 region of the alpha-globin locus on human chromosome 16 was detected as part of an effort to better define the transcriptional profiles of human reticulocytes. Cloning and sequencing of that transcript (GenBank AY698022; named mu-globin) revealed an insert with a 423-nucleotide open reading frame. BLASTP and ClustalW and phylogenetic analyses of the predicted protein demonstrated a high level of homology with the avian alpha-D globin. In addition, the heme- and globin-binding amino acids of mu-globin and avian alpha-D globin are largely conserved. Using quantitative real-time polymerase chain reaction (PCR), mu-globin was detected at a level of approximately 0.1% that measured for alpha-globin in erythroid tissues. Erythroid-specific expression was detected by Northern blot analysis, and maximal expression during the erythroblast terminal differentiation was also detected. Despite this highly regulated pattern of mu-globin gene transcription, mu-globin protein was not detected by mass spectrometry. These results suggest the human genome encodes a previously unrecognized globin member of the avian alpha-D family that is transcribed in a highly regulated pattern in erythroid cells.

Advancing cancer biotherapy with proteomics.

Proteomics is becoming increasingly important for cancer biotherapy. The development of high-throughput platforms now allows the analysis of multiple proteins from small quantities of material. While these techniques are being used to discover new biomarkers, they are particularly important for assessing complex biologic processes such as immunotherapy for cancer. Recent advances in this field are reviewed, as well as the use of proteomics to assess the effectiveness and toxicities of high-dose IL-2 cancer therapy. Proteomics is becoming useful in assessing cancer biotherapies and in unraveling their mechanisms of action. High-throughput proteomic technologies have now advanced to a stage where they have the potential to become effective discovery tools for biomarkers/predictors of disease, disease recurrence, and response to therapy.

Peptide profiling in epithelial tumor plasma by the emerging proteomic techniques.

The plasma peptide component (PPC) from ten melanoma (Mel), breast cancer (BC) and healthy individuals was examined by a combination of RP-HPLC, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and tandem mass spectrometry. A three peak pattern (2023, 2039, 2053.5 m/z) was primarily observed in melanoma. Two peaks (2236.1 and of 2356.3 m/z) were found only in BC samples. Fibrinogen alpha and inter-alpha-trypsin inhibitor heavy chain H4 fragments were absent in both tumor samples.