Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

By means of sensitive and specific methods for histochemical and biochemical determination of dopamine (DA), noradrenaline (NA) and 5‐hydroxytryptamine (5‐HT) we have succeeded in demonstrating and mapping out a number of ascending monoamine neuron systems from the lower brain stem by studying the anterograde and retrograde changes that occur in these neurons after various types of brain lesions. In this way it has been possible to discover: 1) a large, uncrossed nigro‐neostriatal DA neuron system; 2) a DA neuron system, arising from cell‐bodies in the mesen‐cephalon, ascending uncrossed in the medial forebrain bundle close to the nigro‐neostriatal DA fibres, and innervating e.g. the tuberculum olfactorium and nuc. accumbens; 3) ascending NA neuron systems with cell‐bodies situated mainly in the medulla oblongata and pons (locus coeruleus, formatio reticularis), and axons running uncrossed mainly in the medial forebrain bundle, innervating e.g. the limbic forebrain structures, the neocortex and the hypothalamus; 4) ascending 5‐HT' neurons with cell‐bodies situated mainly in the raphe nuclei of the mesencephalon (nuc. raphe dorsalis, nuc. raphe medianus), and axons running uncrossed mainly in the medial forebrain bundle, innervating e.g. the limbic forebrain structures and the hypothalamus. The effects observed on the amine levels of the neurons represent intraneuronal and not transsynaptic changes.

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A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra IIe at position 65. Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.

Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

Summary. Endothelial cell membrane-bound thrombomodulin (TM) plays a critical role as a cofactor in the protein C pathway, important in regulating coagulation as well as inflammation. Heterogeneous soluble TM fragments circulate in the plasma and are found at increased levels in various diseases such as cardiovascular disease and diabetes, and in ischemic and/or inflammatory endothelial injuries. The anticoagulant function of these soluble fragments has not been measured in healthy individuals or in patients. Using an immobilized monoclonal antibody against TM and a microtiter plate format, an assay was designed to capture the soluble TM fragments in plasma and measure their cofactor activity in the thrombin-mediated activation of protein C. In addition, soluble TM antigen levels were measured by enzyme-linked immunosorbent assay. Both assays were used to investigate a group of healthy blood donors. TM fragments released into plasma were shown to retain significant cofactor activity, and reference intervals for healthy men and women were established. Furthermore, a statistically significant correlation was observed between soluble TM antigen levels and soluble TM cofactor activity. This notwithstanding, soluble TM activity only accounted for a minor part of all variation in soluble TM antigen levels (R 2 ¼ 22% in men and R 2 ¼ 16% in women).

The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/ liter. This level was notably low compared with similar products containing identical C H 1, C L , and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in V L increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 3 Ser, Thr-45 3 Lys, Thr-77 3 Ser, and Leu-78 3 Val were most powerful. In addition, replacing four V H residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumorassociated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli.

Three different size classes of cDNA clones coding for the &subunit of human alcohol dehydrogenase (ADH) were characterized from a human liver cDNA library. Clones were identified by hybridization with synthetic oligodeoxyribonucleotides.A total of 2530 nucleotides were determined, covering an ADH-coding region of 1122 nucleotides, a preceding 72-nucleotide segment and 3 types of 3'-non-coding region. The coding nucleotide sequence is in full agreement with the amino acid sequence of the &subunit. Of 8 clones identified, 6 had a short, 213-nucleotide 3'-non-coding region; 1 an intermediate, 590-nucleotide 3'-region; and 1 a long, 1330-nucleotide 3'-region. In addition, 2 unused polyadenylation signals were found. These results suggest that human liver /I-ADH mRNAs occur in several size classes, and that in addition to the consensus sequence AATAAA further signals are important for 3'-end formation.Alcohol dehydrogenase cDNA