Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C₁₈ reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C₁₈ ODS Hypersil analytical column (5 μm particle size; 250 mm × 4.6 mm I.D.) using an acetonitrile—water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N′,N′-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75–400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.     

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

Summary Proteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.Supported by grant Ki 154/6-7 from the Deutsche Forschungsgemeinschaft     

Interpretable pairwise distillations for generative protein sequence models

Many different types of generative models for protein sequences have been proposed in literature. Their uses include the prediction of mutational effects, protein design and the prediction of structural properties. Neural network (NN) architectures have shown great performances, commonly attributed to the capacity to extract non-trivial higher-order interactions from the data. In this work, we analyze two different NN models and assess how close they are to simple pairwise distributions, which have been used in the past for similar problems. We present an approach for extracting pairwise models from more complex ones using an energy-based modeling framework. We show that for the tested models the extracted pairwise models can replicate the energies of the original models and are also close in performance in tasks like mutational effect prediction. In addition, we show that even simpler, factorized models often come close in performance to the original models.     

Subcellular dynamics and functional activity of the cleaved intracellular domain of the Na+ channel β1 subunit

The voltage-gated Na⁺ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na⁺ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD is necessary and sufficient to increase Na⁺ current measured at the plasma membrane. Importantly, although the endogenous Na⁺ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Na_v1.5), the Na⁺ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Finally, we found β1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Na_v1.1 and SCN9A/Na_v1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer.     

Ceramide levels in blood plasma correlate with major depressive disorder severity and its neutralization abrogates depressive behavior in mice

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis that will affect ∼10% of people during their lifetime. Therapy for MDD requires prolonged treatment and often fails, predicating a need for novel treatment strategies. Here, we report increased ceramide levels in the blood plasma of MDD patients and in murine stress-induced models of MDD. These blood plasma ceramide levels correlated with the severity of MDD in human patients and were independent of age, sex, or body mass index. In addition, intravenous injection of anti-ceramide antibodies or neutral ceramidase rapidly abrogated stress-induced MDD, and intravenous injection of blood plasma from mice with MDD induced depression-like behavior in untreated mice, which was abrogated by ex vivo preincubation of the plasma with anti-ceramide antibodies or ceramidase. Mechanistically, we demonstrate that ceramide accumulated in endothelial cells of the hippocampus of stressed mice, evidenced by the quantitative measurement of ceramide in purified hippocampus endothelial cells. We found ceramide inhibited the activity of phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus. Finally, we show intravenous injection of PLD or phosphatidic acid abrogated MDD, indicating the significance of this pathway in MDD pathogenesis. Our data indicate that ceramide controls PLD activity and phosphatidic acid formation in hippocampal endothelial cells and thereby mediates MDD. We propose that neutralization of plasma ceramide could represent a rapid-acting targeted treatment for MDD.     

Infection intensity-dependent accuracy of reagent strip for the diagnosis of Schistosoma haematobium and estimation of treatment prevalence thresholds

BackgroundReagent strip to detect microhematuria as a proxy for Schistosoma haematobium infections has been considered an alternative to urine filtration for individual diagnosis and community-based estimates of treatment needs for preventive chemotherapy. However, the diagnostic accuracy of reagent strip needs further investigation, particularly at low infection intensity levels.MethodsWe used existing data from a study conducted in Tanzania that employed urine filtration and reagent strip testing for S. haematobium in two villages, including a baseline and six follow-up surveys after praziquantel treatment representing a wide range of infection prevalence. We developed a Bayesian model linking individual S. haematobium egg count data based on urine filtration to reagent strip binary test results available on multiple days and estimated the relation between infection intensity and sensitivity of reagent strip. Furthermore, we simulated data from 3,000 hypothetical populations with varying mean infection intensity to infer on the relation between prevalence observed by urine filtration and the interpretation of reagent strip readings.Principal findingsReagent strip showed excellent sensitivity even for single measurement reaching 100% at around 15 eggs of S. haematobium per 10 ml of urine when traces on reagent strip were considered positive. The corresponding specificity was 97%. When traces were considered negative, the diagnostic accuracy of the reagent strip was equivalent to urine filtration data obtained on a single day. A 10% and 50% urine filtration prevalence based on a single day sampling corresponds to 11.2% and 48.6% prevalence by reagent strip, respectively, when traces were considered negative, and 17.6% and 57.7%, respectively, when traces were considered positive.Conclusions/SignificanceTrace results should be included in reagent strip readings when high sensitivity is required, but excluded when high specificity is needed. The observed prevalence of reagent strip results, when traces are considered negative, is a good proxy for prevalence estimates of S. haematobium infection by urine filtration on a single day.     

Interspecific patterns for egg and clutch sizes of African Bufonidae (Amphibia: Anura)

Little is known about reproductive trade-offs in African amphibians, but such data, particularly in the form of quantitative measurements, are a key for investigating life history evolution. Here we compile and analyze known data on African bufonids from published material and new data from preserved museum specimens, to investigate interspecific patterns of egg and clutch sizes variation. Our data is a composite of mixed sources, including ova data from dissected females and laid clutches from observations in the field. Our study shows that, as body size increases, clutch size increases but egg size decreases, and when correcting for body size, egg size is inversely correlated with clutch size. These parameter interactions however, are different for different reproductive modes. In free-swimming larval developing species, the same trends are recovered, but for lecithotrophic viviparous species no significant correlations could be recovered for clutch size and body size nor for the trade-off between clutch size and egg size, and egg size is positively related to body size. The egg size of Nimbaphrynoides occidentalis (Angel, 1943) is a clear outlier, which may be due to its matrotrophic viviparous reproduction. In addition, we observed no statistical difference between ova data collected from dissections and laid clutch data from field observations, which suggests that such a mixed dataset has utility in comparative analyses.