Discovery and development of next generation sGC stimulators with diverse multidimensional pharmacology and broad therapeutic potential.

Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC), an enzyme that catalyzes the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophophate (cGMP), transduces many of the physiological effects of the gasotransmitter NO. Upon binding of NO to the prosthetic heme group of sGC, a conformational change occurs, resulting in enzymatic activation and increased production of cGMP. cGMP modulates several downstream cellular and physiological responses, including but not limited to vasodilation. Impairment of this signaling system and altered NO-cGMP homeostasis have been implicated in cardiovascular, pulmonary, renal, gastrointestinal, central nervous system, and hepatic pathologies. sGC stimulators, small molecule drugs that synergistically increase sGC enzyme activity with NO, have shown great potential to treat a variety of diseases via modulation of NO-sGC-cGMP signaling. Here, we give an overview of novel, orally available sGC stimulators that Ironwood Pharmaceuticals is developing. We outline the non-clinical and clinical studies, highlighting pharmacological and pharmacokinetic (PK) profiles, including pharmacodynamic (PD) effects, and efficacy in a variety of disease models.

A Case of Successful Thromboelastographic Guided Resuscitation after Postpartum Hemorrhage and Cardiac Arrest.

Amniotic fluid embolism (AFE) is an unusual cause of life threatening peri partum hemorrhage (PPH). AFE resuscitation is often associated with renal and respiratory insufficiency, and a coagulopathy similar to disseminated intravascular coagulation (DIC). Resuscitation requires immediate recognition and limited use of crystalloid. We present a case of PPH caused by AFE with resultant cardiac arrest, renal and respiratory failure, and DIC-like coagulopathy, whose successful resuscitation was guided by perfusionist-directed serial thromboelastography (TEG). Viscoelastic tests (VET)s, including the TEG and rotational thromboelastometry (ROTEM), may provide more individualized blood component therapy (BCT) in the treatment of severe PPH associated with AFE as has been previously noted with trauma resuscitation in the literature. However, VET's efficacy is often limited by a lack of standardization, quality assurance norms, and consistent operator proficiency. We suggest that there may be a role for perfusionsts adept at utilizing TEG in the optimization of BCT and adjunctive hemostatic agents in severely hemorrhagic patients. This patient's successful resuscitation demonstrates the importance of resuscitation guided by the perfusionist or other medical professionals with expertise in TEG guided resuscitation and how the administration of specific blood products and hemostatic agents guided by the TEG can help optimize patient outcomes in comparison to traditional 1:1:1 packed red blood cells (PRBC) /fresh frozen plasma (FFP) /platelets ratios given to severely hemorrhaging patients.

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice.

AIMS/HYPOTHESIS: Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice. METHODS: Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER(T2) mice crossed with Ptp1b floxed (Ptp1b(fl/fl)) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b(-/-) mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined. RESULTS: Despite no significant change in body weight relative to HFD-fed Ptp1b(fl/fl) control mice, HFD-fed SA-Ptp1b(-/-) mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase 2 phosphorylation, and decreased expression of Pepck. CONCLUSIONS/INTERPRETATION: Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects.

Low energy prepulse for 10 Hz operation of a soft-x-ray laser.

The influence on Nickel-like Molybdenum soft-x-ray laser performance and stability of a low energy laser prepulse arriving prior to the main laser pumping pulses is experimentally investigated. A promising regime for 10 Hz operation has been observed. A four times increase in soft-x-ray laser operation time with a same target surface is demonstrated. This soft-x-ray laser operation mode corresponds to an optimum delay between the prepulse and the main pulses and to a prepulse energy greater than 20 mJ. We also show that this regime is not associated with a weaker degradation of the target or any reduced ablation rate. Therefore the role of preplasma density gradient in this effect is discussed.

Plasma temperature measurement with a silicon photomultiplier (SiPM).

The temperature of a nonneutral plasma confined in a Penning-Malmberg trap can be determined by slowly lowering one side of the trap's electrostatic axial confinement barrier; the temperature is inferred from the rate at which particles escape the trap as a function of the barrier height. In many experiments, the escaping particles are directed toward a microchannel plate, and the resulting amplified charge is collected on a phosphor screen. The screen is used for imaging the plasma but can also be used as a Faraday cup (FC) for a temperature measurement. The sensitivity limit is then set by microphonic noise enhanced by the screen's high-voltage bias. Alternately, a silicon photomultiplier (SiPM) can be employed to measure the charge via the light emitted from the phosphor screen. This decouples the signal from the microphonic noise and allows the temperature of colder and smaller plasmas to be measured than could be measured previously; this paper focuses on the advantages of a SiPM over a FC.

Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation.

We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms.

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains.

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].

Mutagenicity of the antitumor antibiotic CC-1065 and its analogues in mammalian (V79) cells and bacteria.

CC-1065 is a very potent antitumor antibiotic which binds in the minor groove of DNA with alkylation at N-3 of adenine. Since CC-1065 caused delayed deaths in mice at therapeutic doses, analogues were prepared whose antitumor and biochemical activities have been reported. In this study, the mutagenicity for V79 cells (6-thioguanine resistance) and Salmonella (histidine auxotrophy or azaguanine resistance) of selected analogues was compared to DNA-binding activity and the structure-activity relationship was determined. CC-1065, U-62,736, U-66,866, U-66,694, U-67,786, and U-68,415 all have an A segment with an intact cyclopropyl group and different B segments. The cyclopropyl group is absent from U-66,226 and U-63,360. Elimination of the cyclopropyl ring diminished the cytotoxic and mutagenic potency of the compounds such that U-63,360 was nearly three orders of magnitude less potent than CC-1065 in V79 cells. For the compounds with an intact cyclopropyl group, the order of cytotoxic and mutagenic potency (molar basis) in V79 cells generally correlated with binding to calf thymus DNA, and increased with the length of the B segment. Thus, the order of cytotoxicity was CC-1065 greater than U-68,415 greater than U-66,694 greater than U-66,866 greater than U-62,736. U-67,786 fell outside this pattern since it was more cytotoxic and mutagenic than U-66,694, although it was of a similar size and had similar DNA-binding activity. These results show that an electrophilic carbon afforded by an intact cyclopropyl group of this type is necessary but not sufficient to account for the high cytotoxic and mutagenic potency of CC-1065 and U-68,415. The size and characteristics of the B segment also affect the potency. At an equitoxic (10 or 50% lethal dose) dose, an inverse relationship exists between cytotoxic and mutagenic potency such that at the 50% lethal dose, the least cytotoxic compound (U-62,736) was more mutagenic than the most cytotoxic compound (CC-1065). We speculate that the more cytotoxic analogues are less mutagenic (at an equitoxic dose) because they may have greater structure-directed binding to less mutable DNA sites in the minor groove.

Comparative genotoxicity of adriamycin and menogarol, two anthracycline antitumor agents.

Adriamycin and menogarol are anthracyclines which cause more than 100% increase in life span of mice bearing P388 leukemia and B16 melanoma. Unlike Adriamycin, menogarol does not bind strongly to DNA, and it minimally inhibits DNA and RNA synthesis at lethal doses. Adriamycin is a clinically active drug, and menogarol is undergoing preclinical toxicology at National Cancer Institute. In view of the reported mutagenicity of Adriamycin, we have compared the genotoxicity of the two drugs. Our results show that, although Adriamycin and menogarol differ significantly in their bacterial mutagenicity (Ames assay), they have similar genotoxic activity in several mammalian systems. Adriamycin is strongly mutagenic in the Ames assay with TA98 and TA100. Menogarol is nonmutagenic to TA98 and TA100. For the mammalian cell culture systems, V79 (Chinese hamster) cells are exposed for 2 hr to drug, following which cell survival, induction of sister chromatid exchanges, chromosome damage, and production of mutants resistant to 6-thioguanine are measured. The percentage of survival obtained with the two drugs ranges between 25 and 50% at 0.15 microgram/ml and 5 to 15% at 0.3 microgram/ml. At 0.15 microgram/ml, Adriamycin and menogarol increase the percentage of cells with chromosome damage from a background level of 8.8 to 30 and 22.5%, respectively. The same drug concentration causes a small but significant increase in sister chromatid exchange rate. Both drugs are equally active (increase mutation frequency about 3- to 6-fold above background) in producing 6-thioguanine-resistant mutants. The induction of micronuclei in polychromatic erythrocytes of rats is the most sensitive assay system. Both drugs cause 10- to 15-fold increase in micronuclei at nontoxic doses.

Characterization of sequence elements involved in p53 stability regulation reveals cell type dependence for p53 degradation.

The growth suppressive properties of the tumor suppressor protein p53 are activated upon DNA damage. The activation of p53 is reflected in increased p53 levels which are, at least in part, the result of an extended half-life of the protein. Although this suggests that stabilization of p53 is an intrinsic feature of p53 activation, the mechanisms involved in p53 degradation and stabilization are poorly understood. Here we report on the identification of an internal deletion mutant of wild-type p53, termed delta62-96, which can be stably expressed in various cell lines. This deletion mutant has a turnover rate similar to wild-type p53 and its stability is upregulated by treatment with UV light. In cell lines that express endogenous mutant or no p53, however, delta62-96 appears to be stable, strongly indicating that these cell lines have lost the ability to degrade p53. Further characterization of delta62-96 by mutational analyses defines sequence and structural requirements for p53 degradation and indicates that none of the known p53 phosphorylation sites is essential with respect to p53 stability regulation upon UV-irradiation.

Nucleotide homologies in genes encoding members of the S100 protein family.

Members of the S100 protein family exhibit a unique pattern of cell/tissue-specific expression and approx. 50% similarity at the amino-acid level. The cDNAs encoding many of these proteins from a variety of species are now available making a comparison of these family members at the nucleotide level possible. With few exceptions, family members exhibited less nucleotide identity than amino-acid similarity. Furthermore, the pattern of divergence calculated on the basis of nucleotide identity did not always agree with that calculated on the basis of amino-acid similarity. The majority of sequence diversity occurred in the nontranslated regions suggesting that these regions may be involved in directing the expression of particular members of the family to specific cell types. When comparisons of individual family members were made across species, the following order of species diversity was observed: rat/mouse < human/bovine < porcine < rabbit/avian < Xenopus laevis. The structure of the gene loci encoding these proteins was remarkably conserved both within family members of a given species as well as in individual family members from different species. Although there appears to be great diversity in the 5' flanking regions of these genes, members of the family share at least one common potential regulatory element-the S100 protein element. Thus, membership in the S100 family could be ascertained on the basis of gene organization and the presence of an SPE. Although functional data are limited, the available data indicate that the regulation of the expression of S100 family members is complex and involves both positive and negative regulatory elements. Additional nucleic acid sequences and complimentary functional studies will be required to dissect the mechanisms which target the expression of the members of this family to specific cell types during development.

The role of cysteine residues in S100B dimerization and regulation of target protein activity.

Previous studies have demonstrated that the two cysteine residues in the calcium-binding protein S100B are required for its extracellular functions. In the present study, a recombinant S100B protein and mutant S100Bs containing one or no cysteine residue(s) have been used to determine the contribution of cysteine residues to S100B dimerization and interaction with the intracellular target proteins aldolase, phosphoglucomutase, and the microtubule associated tau protein. Mutation of C68 to a valine or C84 to a serine, C68 to valine and C84 to serine, or C68 to valine and C84 to alanine did not significantly alter S100B activation of aldolase. However, mutation of C84 to serine resulted in calcium-independent S100B activation of phosphoglucomutase and a loss of S100B inhibition of tau phosphorylation by Ca2+/calmodulin-dependent protein kinase II. The altered functionality of the C84S mutant with phosphoglucomutase and tau was not due to altered physical properties or dimerization state. All of the mutants exhibited heat stability and calcium dependent conformational changes which were identical to recombinant S100B. In addition, S100B proteins containing two, one or no cysteine residues behaved as dimers in size exclusion chromatography experiments in the presence or absence of calcium as well as in the presence or absence of reducing agent. Dynamic light scattering and analytical ultracentrifugation experiments confirmed that dimerization was not affected by calcium or reducing agent. Altogether these results demonstrate that S100B dimerization is not calcium- or sulfhydryl-dependent. In summary, cysteine residues are not necessary for the noncovalent dimerization of S100B, but are important in certain S100B target protein-interactions.

S100-mediated signal transduction in the nervous system and neurological diseases.

This article presents new information regarding the complement/level of S100 family members expressed in the brain and reviews the contribution of brain S100 family members to nervous system function and disease. A total of ten S100 family members are reported in the literature to be expressed in brain -S100A1, S100A2, S100A4, S100A5, S100A6, S100A10, S100A11, S100A13, S100B, and S100Z. Quantitative Northern blot analysis detected no S100A3, S100A8, S100A9 or S100A14 mRNA in mouse brain suggesting that these family members are not expressed in the brain. In addition, there was a 100-fold range in the mRNA levels for the six family members that were detected in mouse brain: S100A1/S100B levels were 5-fold higher than S100A6/S100A10 levels and 100-fold higher than S100A4/S100A13 levels. Five of these six family members (S1100A1, S100A6, S100A10, S100A13, and S100B) exhibited age-dependent increases in expression in adult mice that ranged from 5- to 20-fold. Although previous studies on S100 function in the nervous system have focused on S100B, other family members (S100A1, S100A3, S100A4, S100A5) have been implicated in neurological diseases. Like S100B, intra- and inter-cellular forms of these family members have been linked to cell growth, cell differentiation, and apoptotic pathways. Studies presented here demonstrate that ablation of S100A1 expression in PC12 cells results in increased resistance to Abeta peptide induced cell death, stabilization of intracellular [Ca2+] homeostasis, and reduced amyloid precursor protein expression. Altogether, these results confirm that S100-mediated signal transduction pathways play an important role in nervous system function/disease and implicate S100A1 in the neuronal cell dysfunction/death that occurs in Alzheimer's disease.

Glucose and amino acid turnover in untreated gestational diabetes.

OBJECTIVE: Although gestational diabetes affects as many as 3% of all pregnant women, specific aspects of glucose and protein metabolism in this population have not been clearly delineated. We tested the hypothesis that gestational diabetes mellitus (GDM) results in increased glucose production and proteolysis during fasting. RESEARCH DESIGN AND METHODS: Using tracer isotope infusions, the rate of appearance (Ra) of glucose, leucine, phenylalanine and tyrosine, phenylalanine hydroxylation, leucine oxidation, and urea nitrogen excretion were determined after an overnight fast in 10 GDM subjects, within 2 weeks of diagnosis and before initiation of treatment, and in a matched control group of nine healthy nondiabetic pregnant women. RESULTS: Fasting glucose Ra was similar in GDM patients and control subjects (GDM, 12.8 +/- 1.1 vs. control subjects, 12.8 +/- 0.9 mumol . kg-1 . min-1). Leucine and phenylalanine Ra (reflecting proteolysis) also were not different between GDM patients and control subjects (GDM leucine Ra, 128 +/- 14 vs. control subjects, 124 +/- 5; phenylalanine Ra GDM, 35 +/- 4 vs. control subjects, 40 +/- 2 mumol . kg-1 . h-1). Furthermore, leucine oxidation and phenylalanine hydroxylation were not increased in GDM subjects, urea nitrogen excretion was actually lower in GDM patients. However, fasting insulin concentrations were significantly elevated in GDM subjects (GDM, 165 +/- 35 vs. control subjects, 30 +/- 5 pmol/l; P < 0.01). CONCLUSIONS: Hepatic glucose release and whole-body proteolysis in GDM patients were remarkably similar to matched pregnant control subjects. This was achieved with insulin concentrations three- to fivefold higher than normal, suggesting significant insulin resistance for both glucose and protein metabolism in GDM.

Differential regulation of A gamma and G gamma fetal hemoglobin mRNA levels by hydroxyurea and butyrate.

In clinical studies, both hydroxyurea and butyrate increase fetal hemoglobin expression and ameliorate the symptoms of sickle cell anemia. However, comparative studies of the effects of hydroxyurea and butyrate on the expression of the individual fetal hemoglobin genes, A gamma and G gamma, have not been performed. The present study reports the effects of hydroxyurea and butyrate on steady-state A gamma and G gamma mRNA levels in K562 cells. Because the high degree of homology between the A gamma and G gamma cDNA sequences precludes the use of large cDNA probes for detection of individual fetal hemoglobin gene products, we investigated the specificity of two 20-base oligonucleotide probes synthesized from the region of greatest sequence diversity between these genes. Hybridization experiments demonstrated that the A gamma oligonucleotide probe was specific for A gamma DNA and RNA sequences and the G gamma oligonucleotide probe was specific for G gamma DNA and RNA sequences. These oligonucleotide probes detected both A gamma and G gamma mRNAs in K562 cells. In K562 cells treated with 2 mM sodium butyrate for 168 hours, the G gamma mRNA level increased 3.6-fold, whereas the A gamma mRNA level was not significantly different from untreated cells. Similar results were obtained when K562 cells were treated with 80 microM hydroxyurea. The G gamma mRNA level increased 2.3-fold at 168 hours, whereas the A gamma mRNA level did not change. The above results demonstrate that both butyrate and hydroxyurea selectively increase G gamma expression. Selective regulation of individual fetal hemoglobin genes is also seen in human development, where approximately 70% of the total fetal hemoglobin in the fetus is G gamma. Therefore, understanding the mechanisms by which butyrate and hydroxyurea differentially regulate fetal hemoglobin gene expression may provide insights into the developmental regulation of hemoglobin expression as well as the mechanisms of action of pharmacological agents currently being used to treat sickle cell disease.

Tiered approaches to chromatographic bioanalytical method performance evaluation: recommendation for best practices and harmonization from the Global Bioanalysis Consortium harmonization team.

The A2 harmonization team, a part of the Global Bioanalysis Consortium (GBC), focused on defining possible tiers of chromatographic-based bioanalytical method performance. The need for developing bioanalytical methods suitable for the intended use is not a new proposal and is already referenced in regulatory guidance language. However, the practical implementation of approaches that differ from the well-established full validation requirements has proven challenging. Advances in technologies, the need to progress drug development more efficiently, and emerging new drug compound classes support the use of categorized tiers of bioanalytical methods. This paper incorporated the input from an international team of experienced bioanalysts to surmise the advantages and the challenges of tiered approaches and to provide recommendations on paths forward.

Identification of an S100 target protein: glycogen phosphorylase.

An S100 binding protein from skeletal muscle, R95 000, has been purified, identified as glycogen phosphorylase, and shown to be regulated in vitro by the S100 alpha isoform. When a soluble skeletal muscle fraction was subjected to a standard purification procedure for glycogen phosphorylase, R95 000 copurified with the 95 000 molecular weight glycogen phosphorylase protein standard on SDS-polyacrylamide gels, as well as having glycogen phosphorylase activity. In addition, purified glycogen phosphorylase a and b interacted with both S100 isoforms, S100 alpha and S100 beta, by gel overlay and affinity chromatography. While S100 beta had no effect on the enzymatic activity of glycogen phosphorylase a, S100 alpha inhibited the enzymatic activity of glycogen phosphorylase a in a calcium-independent manner. Altogether, these data suggest that glycogen phosphorylase may be an intracellular S100 alpha target in skeletal muscle fibers. Furthermore, these results suggest that the inhibition of glycogen phosphorylase a activity may be responsible for the lack of fatigability of slow-twitch fibers, which express S100 alpha, when compared to fast-twitch fibers, which do not express S100 proteins.

Identification of an S100A1/S100B target protein: phosphoglucomutase.

Phosphoglucomutase was identified as a potential intracellular S100 target protein because it interacted with two members of the S100 family of calcium-modulated proteins, S100A1 and S100B, in gel overlay experiments. These results were confirmed by affinity chromatography experiments demonstrating that S100A1 and S100B bound to phosphoglucomutase-Sepharose in a calcium-dependent manner. In the reverse experiment, phosphoglucomutase bound to S100A1 and S100B-Sepharose in a calcium-dependent manner. S100A1 inhibited phosphoglucomutase activity in a calcium-dependent manner. In contrast, S100B stimulated phosphoglucomutase activity in a calcium-dependent manner. Other calcium-binding proteins (calmodulin, troponin C, parvalbumin, and alpha-lactalbumin) had no effect on phosphoglucomutase. These results suggest that the effects of S100A1 and S100B are not nonspecific effects of low molecular weight, acidic proteins. This is the first report of an S100 target protein whose activity is antagonistically regulated by S100A1 and S100B, suggesting that cellular diversity in intracellular calcium signaling pathways may be due, at least in part, to the complement of S100 proteins expressed in different cell types.

S100A1 and S100B expression and target proteins in type I diabetes.

Calcium receptor proteins are an essential link between hormones that alter intracellular calcium levels and the generation of cellular responses. However, there is no information available regarding the role of calcium receptor proteins, in particular the S100 family, in insulin action and/or diabetes. This study examines the effects of streptozotocin-induced type I diabetes on the expression of the individual S100A1 and S100B isoforms as well as their binding proteins. Diabetes did not increase (or initiate) S100B expression in any non-S100B-expressing tissue (skeletal muscle, heart, kidney, liver, spleen, and pancreas). In all S100B-expressing tissues examined (brain, white fat, and testes), S100B protein levels increased approximately 2-fold while steady state S100B messenger RNA (mRNA) levels decreased. S100A1-expressing tissues exhibited increased (kidney and lung), decreased (skeletal muscle), and unchanged (brain and heart) S100A1 protein levels. While noncoordinate changes in S100A1 protein and steady state mRNA levels were observed in heart, other S100A1-expressing tissues (brain, slow twitch skeletal muscle, and kidney) exhibited coordinate changes in S100A1 protein and steady state mRNA levels. Altogether, these results suggest that the effects of diabetes on S100 expression are isoform as well as tissue-specific. Gel overlay analysis of the S100-binding protein profile revealed both increases and decreases in binding proteins in all tissues examined. In summary, changes in the expression of S100A1, S100B, and S100-binding proteins occur in type I diabetes and represent important molecular events in the effects of insulin/insulin insufficiency on cell function.

Detection of SYT-SSX1/2 fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) is a valuable diagnostic tool in synovial sarcoma.

Cytogenetically, most synovial sarcomas are characterised by a specific chromosomal translocation [(X;18) (p11.2;q11.2)], which results in the generation of fusion transcripts comprising SYT (18q11) and either SSX1 or SSX2 (Xp11) sequences. By using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, specific SYT-SSX1/2 fusion transcripts were detected in 10 histopathologically confirmed synovial sarcomas. Control tumours with morphological spindle cell patterns mimicking monophasic synovial sarcoma tested negative (18/19) in the RT-PCR protocol, with the exception of one spindle cell sarcoma originally classified as a fibrosarcoma. Furthermore, the established RT-PCR protocol was used to evaluate the feasibility of SYT-SSX1/2 fusion transcript detection for minimal residual disease analysis. Analyses of surgical margins revealed a fusion transcript in two of four operations for synovial sarcoma analysed, one of which was diagnosed with tumour free margins by conventional histopathology. These data suggest that the RT-PCR amplification of SYT-SSX1/2 fusion transcripts is a valuable tool in the differentiation of synovial sarcomas, especially in cases of equivocal morphology. Additionally, the RT-PCR approach may be used for the detection of residual tumour cells in synovial sarcoma patients.