Recombinant human IL-6 expressed inE. coli undergoes selective N-terminal degradation: Evidence that the protein consists of a stable core and a nonessential flexible N-terminal

A synthetic gene for human interleukin-6 has been expressed inE. coli. The protein has been purified and renatured and has the same activity as natural human IL-6 using the 7TD1 cell proliferation assay. The protein undergoes specific cleavage by a thiol protease, yielding two new N-termini at Arg-9 and His-15. The truncated proteins retain full biological activity. The degradation results in the loss of sharp amide resonances in the¹H-NMR spectrum, and little change to the ultraviolet CD spectrum. Several amino acid type assignments could be made for these sharp amides using a DQF-COSY 2D-NMR experiment. The N-terminal 15 amino acids exist as a flexible, random coil, attached to a central structure.     

Production of 10-ketostearic acid from oleic acid by a strain of Flavobacterium sp. 12-4A

Summary A new Flavobacterium sp., strain 12-4A, produces solely 10-ketostearic acid (KSA) from oleic acid (OA) in growing cultures. Under optimized conditions 14 mg/ml of KSA was produced from 25 mg/ml of OA after 3 days of cultivation at 30°C.     

Collaborative environmental DNA sampling from petal surfaces of flowering cherry <Emphasis Type="Italic">Cerasus</Emphasis> × <Emphasis Type="Italic">yedoensis</Emphasis> ‘Somei-yoshino’ across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus?×?yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.     

Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters

ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H⁺-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters.     

Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species.

Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.