Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

Glucose-Induced Glucagon-Like Peptide 1 Secretion Is Deficient in Patients with Non-Alcoholic Fatty Liver Disease

Background & Aims

The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal peptide hormones regulating postprandial insulin release from pancreatic β-cells. GLP-1 agonism is a treatment strategy in Type 2 diabetes and is evaluated in Non-alcoholic fatty liver disease (NAFLD). However, the role of incretins in its pathophysiology is insufficiently understood. Studies in mice suggest improvement of hepatic steatosis by GLP-1 agonism. We determined the secretion of incretins after oral glucose administration in non-diabetic NAFLD patients.

Methods

N = 52 patients (n = 16 NAFLD and n = 36 Non-alcoholic steatohepatitis (NASH) patients) and n = 50 matched healthy controls were included. Standardized oral glucose tolerance test was performed. Glucose, insulin, glucagon, GLP-1 and GIP plasma levels were measured sequentially for 120 minutes after glucose administration.

Results

Glucose induced GLP-1 secretion was significantly decreased in patients compared to controls (p<0.001). In contrast, GIP secretion was unchanged. There was no difference in GLP-1 and GIP secretion between NAFLD and NASH subgroups. All patients were insulin resistant, however HOMA2-IR was highest in the NASH subgroup. Fasting and glucose-induced insulin secretion was higher in NAFLD and NASH compared to controls, while the glucose lowering effect was diminished. Concomitantly, fasting glucagon secretion was significantly elevated in NAFLD and NASH.

Conclusions

Glucose-induced GLP-1 secretion is deficient in patients with NAFLD and NASH. GIP secretion is contrarily preserved. Insulin resistance, with hyperinsulinemia and hyperglucagonemia, is present in all patients, and is more severe in NASH compared to NAFLD. These pathophysiologic findings endorse the current evaluation of GLP-1 agonism for the treatment of NAFLD.     

Cerebrospinal Fluid Immunoglobulin Kappa Light Chain in Clinically Isolated Syndrome and Multiple Sclerosis

Background

Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.

Methods

KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction (NIND, n = 77), MS (n = 20), viral CNS infections (V-CNS-I, n = 10), neuroborreliosis (NB, n = 17) and other bacterial CNS infections (B-CNS-I, n = 10). Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).

Results

CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count (p<0.001, R = 0.46), CSF CXCL13 levels (p<0.001, R = 0.64) and also intrathecal IgG synthesis (p<0.001, R = 0.74) as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.

Conclusions

Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS.     

Clinical Features,Treatment and Outcome of Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma of the Ocular Adnexa: Single Center Experience of 60 Patients

Background

Orbital marginal zone B-cell lymphoma (OAML) constitutes for the most frequent diagnosis in orbital lymphoma. Relatively little data, however, have been reported in larger cohorts of patients staged in a uniform way and no therapy standard exists to date.

Material and Methods

We have retrospectively analyzed 60 patients diagnosed and treated at our institution 1999–2012. Median age at diagnosis was 64 years (IQR 51–75) and follow-up time 43 months (IQR 16–92). All patients had undergone uniform extensive staging and histological diagnosis was made by a reference pathologist according to the WHO classification.

Results

The majority of patients presented with stage IE (n = 40/60, 67%), three had IIE/IIIE and the remaining 17 stage IVE. Seven patients with IVE had bilateral orbital disease whereas the others showed involvement of further organs. Treatment data were available in 58 patients. Local treatment with radiotherapy (14/58, 24%) or surgery (3/58, 5%) resulted in response in 82% of patients. A total of 26 patients (45%) received systemic treatment with a response rate of 85%. Nine patients received antibiotics as initial therapy; response rate was 38%. Watchful-waiting was the initial approach in 6/58 patients. In total 28/58 patients (48%) progressed and were given further therapy. Median time-to-progression in this cohort was 20 months (IQR 9–39). There was no difference in time-to-progression after first-line therapy between the different therapy arms (p = 0.14). Elevated beta-2-microglobulin, plasmacytic differentiation, autoimmune disorder and site of lymphoma were not associated with a higher risk for progress.

Conclusion

Our data underscore the excellent prognosis of OAML irrespective of initial therapy, as there was no significant difference in time-to-progression and response between local or systemic therapy. In the absence of randomized trials, the least toxic individual approach should be chosen for OAML.     

The structure of a native l-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 ? resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn(2+) ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed.     

Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.