Lmbrd1 expression is essential for the initiation of gastrulation

The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1). This defect is characterized by massive accumulation of free cobalamin in lysosomes and loss of mitochondrial succinyl‐CoA synthesis and cytosolic methionine synthesis. Affected children suffer from heart defects, developmental delay and megaloblastic anemia. LMBRD1 encodes for LMBD1, a predicted lysosomal cobalamin transport protein. In this study, we determine the physiological function of LMBRD1 during embryogenesis by generating Lmbrd1 deficient mice using the Cre/LoxP system. Complete loss of Lmbrd1 function is accompanied by early embryonic death in mice. Whole mount in situ hybridization studies against bone morphogenetic protein 4 and Nodal show that initial formation of the proximal–distal axis is unaffected in early embryonic stages whereas the initiation of gastrulation is disturbed shown by the expression pattern of even skipped homeotic gene 1 and fibroblast growth factor 8 in Lmbrd1 deficient mice. We conclude that intact function of LMBD1 is essential for the initiation of gastrulation.     

Epigenetic Signatures at AQP3 and SOCS3 Engage in Low-Grade Inflammation across Different Tissues

BackgroundElevated levels of C-reactive protein (CRP, determined by a high-sensitivity assay) indicate low-grade inflammation which is implicated in many age-related disorders. Epigenetic studies on CRP might discover molecular mechanisms underlying CRP regulation. We aimed to identify DNA methylation sites related to CRP concentrations in cells and tissues regulating low-grade inflammation.ResultsGenome-wide DNA methylation was measured in peripheral blood in 1,741 participants of the KORA F4 study using Illumina HumanMethylation450 BeadChip arrays. Four CpG sites (located at BCL3, AQP3, SOCS3, and cg19821297 intergenic at chromosome 19p13.2, P ≤ 1.01E-07) were significantly hypomethylated at high CRP concentrations independent of various confounders including age, sex, BMI, smoking, and white blood cell composition. Findings were not sex-specific. CRP-related top genes were enriched in JAK/STAT pathways (Benjamini-Hochberg corrected P < 0.05). Results were followed-up in three studies using DNA from peripheral blood (EPICOR, n = 503) and adipose tissue (TwinsUK, n = 368) measured as described above and from liver tissue (LMU liver cohort, n = 286) measured by MALDI-TOF mass spectrometry using EpiTYPER. CpG sites at the AQP3 locus (significant p-values in peripheral blood = 1.72E-03 and liver tissue = 1.51E-03) and the SOCS3 locus (p-values in liver < 2.82E-05) were associated with CRP in the validation panels.ConclusionsEpigenetic modifications seem to engage in low-grade inflammation, possibly via JAK/STAT mediated pathways. Results suggest a shared relevance across different tissues at the AQP3 locus and highlight a role of DNA methylation for CRP regulation at the SOCS3 locus.     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.     

Repeatability Using Automatic Tracing with Canon OCT- HS100 and Zeiss Cirrus HD-OCT 5000

Background

Optical coherence tomography (OCT), can be used in clinical practice to provide high resolution cross-sectional images of the retina, optic disc and macula structure. These measurements can be useful for early detection, diagnosis, monitoring and treatment guidance for retinal diseases. Therefore, repeatability of measurements in OCT is of great importance.

Methods

Macula and optic disc parameters from the right eye of 30 healthy subjects were obtained twice with the Canon OCT-HS100 and Zeiss Cirrus HD-OCT 5000. Repeatability was evaluated by use of the coefficient of repeatability (CR) and the coefficient of repeatability as a percentage of the mean (CR%), and the obtained measurements were compared between the instruments.

Results

CR% of optic disc parameters ranged between 0.90 and 22.22% and 0.00 and 16.00% with the Canon and Zeiss OCT respectively. For macular parameters CR% ranged between 0.62 and 2.81% and 0.99 and 1.81% with the Canon and Zeiss OCT respectively. No statistical difference could be found when comparing the CR of all macular and disc measurements between the instruments. Compared to our previously published data repeatability has significantly improved with the inclusion of automatic tracking systems with both the Canon and Zeiss OCT.

Conclusion

Automatic tracking function improves repeatability in both Canon OCT-HS100 and Zeiss Cirrus HD-OCT 5000. However, measurements generated by the two instruments are still not interchangeable.     

Isolation and some properties of lysineN 6-hydroxylase fromEscherichia coli strain EN222

Summary Lysine N⁶-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication ofEscherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium sulfate precipitation and fast protein liquid chromatography using Superose 12 and Mono Q columns. The molecular mass as determined by gel filtration was 200 kDa and 50 kDa by SDS/polyacrylamide gel electrophoresis. The enzyme binds 0.79 molecule FAD/50 kDa. The activity of the enzyme is strictly dependent on NADPH. Its properties are similar to other flavoprotein monooxygenases of the EC group 1.14.13.     

Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites

In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host-mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

Cerebral Polyamine Metabolism in Reversible Hypoglycemia of Rat: Relationship to Energy Metabolites and Calcium

Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-microns thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p less than or equal to 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p less than or equal to 0.01 and p less than or equal to 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro effects of pulsed near-ultraviolet laser exposure on human larynx carcinoma cells.

Effects of pulsed near-ultraviolet laser beam on structural characteristics and macromolecular synthesis of carcinoma HEp2 cells were investigated. Laser irradiation damage induced in these eukaryotic cells could be characterized by two development stages: a) a reversible stage with minor morphological damages (1.5 kJ/m2) and 2) an irreversible one, at higher fluences, characterized by cellular membrane damage, necrobiosis and cells detachment from the substrate (4.5 kJ/m2). A. Studies performed referring to macromolecular syntheses of low laser fluences (1.5 kJ/m2)--irradiated HEp2 cells showed the following aspects: a) syntheses inhibiton phase in the first cycles of cellular replication and b) syntheses stimulation phases in the following cycle with total repair of laser-induced molecular lesions. B. At high laser fluences (3-4.5 kJ/m2), metabolic lesions repair was partially or totally blocked after prolonged culturing at 37 degrees C. Ths paper suggests some mechanisms of laser action on macromolecular synthesis and correlates them with morphological changes induced by laser exposure of carcinoma cells.