Heterogeneity of LSD-displacing factors and multiple types of high affinity LSD-binding sites

Heterogeneity of high affinity LSD-binding sites was confirmed by displacement studies with 2-bromo (+)—LSD and with apamin, a peptide neurotoxin. In line with the concept of multiple binding sites, a number of fractions of putative endogenous ligands could be separated from rat brain extract. The LSD-displacing β-fraction was not detectable in tissues lacking high affinity LSD-binding sites. High affinity dopamine- and serotonin-binding was differentially affected by the β-fraction.     

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Endocytosis of junctional cadherins in bovine kidney epithelial (MDBK) cells cultured in low Ca2+ ion medium

The release of intercellular contacts in MDBK cells, initiated by the depletion of Ca2+ ions from the culture medium, results in the endocytotic uptake of membrane vesicles containing specific membrane constituents of the zonula adhaerens (ZA). During this process the junction-derived, endocytosed vesicles remain associated with the ZA plaque components, while the plaque and its attached actin filaments retract as a whole in a ring-like fashion from the plasma membrane, often accumulating, usually in fragments, in the juxtanuclear cytoplasm. Double-label immunofluorescence microscopy with antiplakoglobin and antivinculin has indicated that both plaque proteins colocalize with the hallmark membrane glycoprotein of this junction type, E-cadherin (uvomorulin). When HRP used as a fluid phase marker is applied to the culture medium, simultaneously with the Ca2+ ion-chelator EGTA, numerous HRP-positive vesicles are found in close association with the dislocated plaque material, suggesting that the HRP is contained in the vesicles formed upon EGTA-induced junction splitting. Immunoelectron microscopy with various cadherin-specific antibodies revealed vesicle-associated labeling, confirming the derivation of these plaque-associated vesicles from the ZA. As the desmosome-specific cadherin, desmoglein, is recovered in another type of junction-derived vesicle, which is characterized by its association with a desmoplakin-plaque, we conclude that the membrane domains of both kinds of junction are endocytosed during Ca2+ depletion but stay in different vesicle populations, emphasizing the selective interaction of the specific cadherins with their respective plaque and filament partners.     

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

Transformation of cultured chick lens epithelial cells with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) leads to radical changes in cell shape and interactions. When cultured at the restrictive temperature (42 degrees C), the transformed cells largely retained epithelial morphology and intercellular adherens junctions (AJ), whereas on switch to the permissive temperature (37 degrees C) they rapidly became fibroblastoid, their AJ deteriorated, and cell adhesion molecules (A-CAM) (N-cadherin) largely disappeared from intercellular contact sites. The microfilament system that was primarily associated with these junctions was markedly rearranged on shift to 37 degrees C and remained associated mainly with cell-substrate focal contacts. These apparent changes in intercellular AJ were not accompanied by significant alterations in the cellular content of several junction-associated molecules, including A-CAM, vinculin, and talin. Immunolabeling with phosphotyrosine-specific antibodies indicated that both cell-substrate and intercellular AJ were the major cellular targets for the pp60v-src tyrosine-specific protein kinase. It was further shown that intercellular AJ components serve as substrates to tyrosine kinases also in nontransformed lens cells, because the addition of a combination of vanadate and H2O2--which are potent inhibitors of protein tyrosine phosphatases--leads to a remarkable accumulation of immunoreactive phosphotyrosine-containing proteins in these junctions. This finding suggests that intercellular junctions are major sites of action of protein tyrosine kinases and that protein tyrosine phosphatases play a major role in the regulation of phosphotyrosine levels in AJ of both normal and RSV-transformed cells.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Regulation of β-adrenergic responses during in vitro differentiation of mouse erythroleukemia cells

Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in β-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the β-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor—cyclase coupling is probably due to a major shift in the expression of G_i and G_s regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is G_i (G_sα:G_iα = 1:7). By contrast, G_s predominates in differentiated cells (G_sα:G_iα = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.     

Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

Multiple mRNAs are generated from the chicken lysozyme gene

We have determined the DNA sequence of a 770 by Pst I fragment containing 450 nucleotides of the 5′ flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5′ end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5′ noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5′ termini of the different mRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5′ ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5′ termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5′ end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.