Abstract.A monoclonal antibody (anti-ctsm-1) recognizing exclusively a-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of a-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-ctsm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for a-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 + 5 % in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of a-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-ctsm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, a-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, a-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma, a-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. FinaUy, ~t-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-asm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions. T HOUGH actin is one of the most conserved eukaryotic proteins, it is expressed in mammals and birds as six isoforms characterized by two-dimensional (2D)-PAGE and amino acid sequence analysis (14,49,56,57,59). Four of them represent differentiation markers of muscle tissues and two are found practically in all cells (56,57). Actin isoforms show >90% overall sequence homology, but only 50-60% homology in their 18 NH2-terminal residues (56, 57). The NH2-terminal region of actin appears to be a major antigenic region (2, 5, 34) and may be involved in the interaction of actin with other proteins such as myosin (31).Little is presently known about the function of actin isoforms. It has been shown that the relative proportions of actin isoforms are different in smooth muscles of different organs (12, 47; Skalli, O., J. Vandekerckhove, and G. Gabbiani, manuscript submitted for publication) and change within the same population of smooth muscle cells (SMCs) t during development (23, 24), pathological situations (11, 21), and different culture conditions (35,46,50). In noumuscle cells, a difference in the proportions of 13-and y-cytoplasmic actins has been reported between normal and tumoral T-lympho-1. Abbreviation used in this paper: SMC, smooth muscle cell. cytes (28). All these studies have been performed on cell populations by means ...

No abstract

A general chemical phosphorylation method based on P(II1) chemistry has been developed. The system is demonstrated for the phosphorylation of oligonucleotides, directly after their synthesis on a solid support, and for the 0-phosphorylation of serine, threonine, and tyrosine as well as for a serine-containing peptide.

An ideal vaccine should elicit a long lasting immune response against the natural parasite, both at the T- and B-cell level. The immune response should occur in all individuals and be directed against determinants that do not vary in the natural parasite population. A major problem in designing synthetic peptide vaccines is that T cells generally recognize peptide antigens only in association with one or a few of the many variants of major histocompatibility complex (MHC) antigens. During the characterization of epitopes of the malaria parasite Plasmodium falciparum that are recognized by human T cells, we analysed a sequence of the circumsporozoite protein, and found that synthetic peptides corresponding to this sequence are recognized by T cells in association with many different MHC class II molecules, both in mouse and in man. This region of the circumsporozoite protein is invariant in different parasite isolates. Peptides derived from this region should be capable of inducing T-cell responses in individuals of most HLA-DR types, and may represent good candidates for inclusion in an effective anti-malaria peptide vaccine.

The tetrapeptide H-Arg-Gly-Asp-Ser-OH (1) (RGDS), representing a recognition sequence of fibrinogen for its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3), served as lead compound for the development of highly potent and selective fibrinogen receptor antagonists. Replacement of the N-terminal arginine by p-amidinophenylalanine or the Gly moiety by m-aminobenzoic acid led to compounds which are superior to the lead peptide with regard to activity and selectivity for GP IIb-IIIa vs the closely related vitronectin receptor alpha v beta 3. By random screening [(p-amidinobenzenesulfonamido)ethyl]-p-phenoxyacetic acid derivatives have been identified as fibrinogen receptor antagonists. Further structure-activity relationship studies culminated in the preparation of N-[N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl]-3-phenyl-L- alanine (29h, Ro 43-5054) and [[1-[N-(p-amidinobenzoyl)-L-tyrosyl]-4-piperidinyl]oxy]acetic acid (37f, Ro 44-9883), which exhibit very high activity as platelet aggregation inhibitors (IC50s 0.06 and 0.03 microM, respectively, human PRP/ADP) as well as marked selectivity for GP IIb-IIIa vs alpha v beta 3. Since the activity of 37f in dogs declines according to a two-compartment model with an initial phase having a t1/2 of 8 min and a second phase with a t1/2 of 110 min, this compound is a suitable candidate for the development as iv platelet inhibitor.

A number of biologically relevant 04-phospho-~-tyrosine-containing peptides have been synthesized by either the global phosphorylation of the side-chain-unprotected L-tyrosine moiety in presynthesized resin-bound peptides or alternatively by the incorporation of suitably protected 04-phospho-~-tyrosine building blocks in the continuous-flow method of Fmoc solid-phase peptide synthesis. Different phosphate-protecting groups have been applied.

Since cholecystokinins (CCK8) seems to be the physiological ligand of CCK receptors in the brain, it would be the most suitable probe for the characterization of CCK receptors in radioligand binding studies.[3H]CCKs was synthetized with a specific radioactivity sufficient for the detection of high affinity binding sites.[3H]CCKs binds saturably and reversibly to distinct sites in rat brain and pancreas with nanomolar affinity. While the C-terminal tetrapeptide of CCK is the minimal structure required for nanomolar affinity in the brain, the entire octapeptide sequence is required for binding affinity in pancreas. Desulfated CCKs and several gastrin-I peptides, which are likewise unsulfated, show virtually no affinity to the binding sites in pancreas but high affinity in cerebral cortex. The ligand specificity of the CCK peptides corresponds to their electrophysiological potency in the brain and their stimulation of secretion in pancreas, respectively. Autoradiographically, high densities of [3H]CCKs binding sites were found in cerebral cortex and olfactory bulb, medium levels in nucleus accumbens, hippocampus, dentate ,qrus, and striatum with virtually no labeling in cerebellum. This pattern is similar to the distribution of CCK-like immunoreactivity in the brain. In pancreas, equally high levels of [3H]CCKs labeling were found in the exocrine and endocrine region.[3H]CCKs binding sites differ from those identified previously with [1251]Bolton-Hunter-CCK33 by their sensitivity to guanyl nucleotides in the brain, their ion dependency in the brain and pancreas, and their different autoradiographical localization in some parts of the brain. The distribution of CCK binding sites labeled with [3H]CCKs appears to correlate better with the CCK immunoreactivity than those labeled with [1251]Bolton-Hunter-CCK33. Thus, [3H]CCK, appears to be the radioligand of choice for the investigation of CCK receptors.

Amphipathic peptides can be useful effectors to enhance gene delivery. However, peptide/DNA complexes usually require additional effectors, such as fusogenic lipids, to mediate efficient transfection. Due to weak and/or multiple interactions between the various components of the system, the transfecting complexes are often heterogeneous and unstable in biological fluids. Accordingly, a hybrid molecule resulting from the covalent coupling of an amphipathic, membrane-disturbing peptide to a lipid moiety might create a stable and efficient peptide-based gene transfer system. The present work describes such a novel hybrid molecule, dioleoylmelittin, resulting from the conjugation of dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)propionate] with [Cys1]melittin. Dioleoylmelittin had a lower hemolytic and membrane-disturbing activity than melittin. Size and zeta potential measurements, DNA gel electrophoresis, and electron microscopy showed that dioleoylmelittin, unlike melittin, was able to complex plasmid DNA to form spherical particles with a net positive charge and a diameter between 50 and 250 nm. These particles, prepared at an optimal 10/1 dioleoylmelittin/DNA ratio (w/w), mediated efficient transient transfection of reporter genes in cultured mammalian cells including primary cells. The luciferase activity induced by the dioleoylmelittin/DNA complex was 5-500-fold higher than that induced by a cationic lipid/DNA complex, depending on the cationic lipid and the cell-line. Surprisingly, the presence of 10-50% fetal calf serum during dioleoylmelittin-mediated transfection enhanced 1.5-3-fold gene expression. Dioleoylmelittin represents a new class of efficient peptide-based transfection reagents, especially suited for serum-sensitive cells.