Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23

Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23. We had previously generated a YAC contig in this region extending from UBE1 to the OATL1 locus. In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus. A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval. The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS 6950-DXS6949. In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160. Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci. This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA. These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region. Genetic cross-overs in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports. With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/ TIMP1-DXS1367-ZNF81-DXS.6849-ZNF21-DXSy6616++ +-(OATL1, DXS6950-DXS6949)- WAS-(GATA, DXS1126)-DXS1240-Xcen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The perinatal mortality rate as an indicator of quality of care in international comparisons

Priming and recollection are expressions of human memory mediated by different brain events. These brain events were monitored while people discriminated words from nonwords. Mean response latencies were shorter for words that appeared in an earlier study phase than for new words. This priming effect was reduced when the letters of words in study-phase presentations were presented individually in succession as opposed to together as complete words. Based on this outcome, visual word-form priming was linked to a brain potential recorded from the scalp over the occipital lobe about 450 ms after word onset. This potential differed from another potential previously associated with recollection, suggesting that distinct operations associated with these two types of memory can be monitored at the precise time that they occur in the human brain.     

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p

OBJECTIVES: To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process. METHODS: OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue. RESULTS: Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in 'inflamed' compared with 'non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables. CONCLUSIONS: Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.     

Evaluation of polycaprolactone − poly‐D,L‐lactide copolymer as biomaterial for breast tissue engineering

The potential of the copolymer polycaprolactone‐co‐ poly‐d ,l ‐lactic acid (PCLLA ) as a biomaterial for scaffold‐based therapy for breast tissue engineering applications was assessed. First, the synthesized PCLLA was evaluated for its processability by means of additive manufacturing (AM ). We found that the synthesized PCLLA could be fabricated into scaffolds with an overall gross morphology and porosity similar to that of polycaprolactone. The PCLLA scaffolds possessed a compressive Young's modulus (ca 46 kPa ) similar to that of native breast (0.5 ? 25 kPa ), but lacked thermal stability and underwent thermal degradation during the fabrication process. The PCLLA scaffolds underwent rapid degradation in vitro which was characterized by loss of the scaffolds' mechanical integrity and a drastic decrease in mass‐average molar mass (M _w) and number‐average molar mass (M _n) after 4 weeks of immersion in phosphate buffer solution maintained at 37 °C. The tin‐catalysed PCLLA scaffold was also found to have cytotoxic effects on cells. Although the initial mechanical properties of the PCLLA scaffolds generally showed potential for applications in breast tissue regeneration, the thermal stability of the copolymer for AM processes, biocompatibility towards cells and degradation rate is not satisfactory at this stage. Therefore, we conclude that research efforts should be geared towards fine‐tuning the copolymer synthesizing methods. © 2016 Society of Chemical Industry     

Observation of the dynamics of live cardiomyocytes through a free-running scanning near-field optical microscopy setup

We report the observation of live-cell dynamics by noncontact scanning near-field optical microscopy (SNOM) modified to work with living biological samples that are fully immersed in liquid. We did not use the SNOM setup in strictly near-field conditions (we used 1-mum constant-height mode); however, we could examine the dynamics of rhythmically beating cardiac myocytes in culture with extremely high vertical sensitivity below the nanometric range. We could halt scans at any point to record localized contraction profiles of the cell membrane. We show that the contractions of the organisms changed shape dramatically within adjacent areas. We believe that the spatial dependency of the contractions arises because of the measurement system's ability to resolve the behavior of individual submembrane actin bundles. Our results, combining imaging and real-time recording in localized areas, reveal a new, to our knowledge, noninvasive method for using SNOM setups for studying the dynamics of live biological samples.     

Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.