Assembly of pre-mRNA splicing complex is cap dependent.

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.     

Über die chromotropen Zellen der Nebenniere vom Wasserfrosch

Zusammenfassung Aus dieser neuerlichen Untersuchung der zuerst von Stilling beschriebenen Sommerzellen ergibt sich somit als einzige Übereinstimmung in den sonst widerspruchsvollen Angaben des Schrifttums, daß sie in ihrem Vorkommen nicht auf den Sommer beschränkt sind, weshalb sie von mir nach ihrer färberischen Eigentümlichkeit als chromotrope Zellen bezeichnet werden. Da ihr isoelektrischer Punkt bei etwa p_H 5 liegt, verhalten sie sich gegenüber Farbstoffen nicht ausschließlich (acido-) oxyphil, doch sind sie auch keine Mastzellen oder überhaupt während der Entwicklung veränderte Wanderzellen, sondern eine besondere Art autochthon entstandener Nebennieren-Epithelzellen, deren Körnchen saure Polysaccharide enthalten. Ihre funktionelle Bedeutung muß erst geklärt werden. Dabei ist es besonders bemerkenswert, daß sich das Vorkommen dieser Zellen auf Rana esculenta und eine Abart von ihr sowie exotische Verwandte beschränkt, während sie bei Rana temporaria und deren nächsten Verwandten immer fehlen. Die gegenteiligen Angaben der Literatur beruhen wahrscheinlich teilweise auf unzutreffender Bestimmung der Art und im übrigen wohl auf Verwechslung mit gekörnten Wanderzellen, was besonders bei der Entwicklung zu falschen Vorstellungen führen kann.Herrn Professor Alfred Kohn in dankbarer Erinnerung gewidmet.     

15N nmr spectroscopy. I. Polysarcosine and related sequence polypeptides

The natural abundance ¹⁵N nmr spectra of linear polysarcosine (DP = 35) has been recorded in Me₂SO and H₂O solution. Because of cis/trans isomerization at the peptide bond, a broad signal with several splittings was observed. These splittings appear to reflect the influence of three peptide bonds on a single N atom. The ¹⁵N signals from the sequence polypeptides (β-Ala-Sar-Gly)_n and (β-Ala-Sar-D ,L -Ala)_n also show a cis/trans splitting, as well as chemical shifts which are dependent on the peptide sequence. The tertiary nitrogen of the sarcosyl residue has a T₁ relaxation time which is longer than the T₁ for secondary nitrogens of the other amino acids. The nuclear Overhauser effect is also discussed.     

Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.     

Oxidative stress‐induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson''s disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4‐TRPML1‐ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca²⁺ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4‐TRPML1‐ALG2 pathway was also activated by H₂O₂, indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation‐induced lysosomal retrograde transport involved both the TMEM55B‐JIP4 and TRPML1‐ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy‐inducing signal.     

Cysteine restriction‐specific effects of sulfur amino acid restriction on lipid metabolism

Decreasing the dietary intake of methionine exerts robust anti‐adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti‐adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine‐ and cysteine‐titrated diets, we demonstrate that the anti‐adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non‐glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck‐M. In mice, the magnitude of SAAR‐induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age‐at‐onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross‐sectional epidemiological study. Controlled feeding of low‐SAA, high‐polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.     

Altered expression of NCAM in hippocampus and cortex may underlie memory and learning deficits in rats with streptozotocin-induced diabetes mellitus

Neurological and structural changes are paralleled by cognitive deficits in diabetes mellitus. The present study was designed to evaluate the expression of neural cell adhesion molecules (NCAM) in the hippocampus, cortex and cerebellum and to examine cognitive functions in diabetic rats. Diabetes was induced in male albino rats via intraperitoneal streptozotocin injection. Learning and memory behaviors were investigated using a passive avoidance test and a spatial version of the Morris water maze test. NCAM expression was detected in the hippocampus, cortex and cerebellum by an immunoblotting method. The diabetic rats developed significant impairment in learning and memory behaviours as indicated by deficits in passive avoidance and water maze tests as compared to control rats. Expression of NCAM 180 and 120 kDa were found to be higher in hippocampus and cortex of diabetic rat brains compared to those of control, whereas expression of NCAM 140 kDa decreased in these brain regions. Our findings suggest that streptozotocin-induced diabetes impairs cognitive functions and causes an imbalance in expression of NCAM in those brain regions involved in learning and memory. Altered expression of NCAM in hippocampus may be an important cause of learning and memory deficits that occur in diabetes mellitus.     

Turkish graveyards as refuges for orchids against tuber harvest

Harvest of orchid tubers for salep production is widespread in southwestern Asia and the Balkans and constitutes a major conservation risk for wild orchid populations. Synanthropic habitats, such as graveyards, are important refuges for orchids and other organisms and could offer protection from salep harvesting because of their special cultural role. However, little is known about the occurrence and factors influencing harvesting of salep in graveyards. During field surveys of 474 graveyards throughout Turkey, we observed 333 graveyards with orchids, 311 graveyards with tuberous orchids, and salep harvest in 14 graveyards. Altogether, 530 individuals of 17 orchid species were collected, representing 9% of the individuals recorded. Harvesting intensity was relatively low, and populations were usually not wholly destroyed. However, some species were clearly more affected than others. Salep harvesting risk of orchid species was significantly associated with flowering time, with early‐flowering species being more affected. A marginally significant positive relationship between harvesting risk and species‐specific tuber size was also detected. Our data suggest that graveyards might offer some protection against salep harvesting in Turkey, but they also show that some orchid taxa are much more affected than others. Overall, our observations add more weight to the conservation value of these special habitats.