Ordered Arrays of Type B Botulinum Toxin On Phospholipid Vesicles

1990 ◽  
Vol 48 (1) ◽  
pp. 496-497
Author(s):  
F. Schmid ◽  
B. R. DasGupta ◽  
J. P. Robinson
Keyword(s):  
Neural Cells ◽  
Disulfide Linkage ◽  
Cell Internalization ◽  
Infection Models ◽  
Botulinum Neurotoxins ◽  
A Cell ◽  
Single Polypeptide ◽  
Endogenous Proteases ◽  
Carboxy Terminal ◽  
Polypeptide Chains

Botulinum neurotoxins comprise a family of potent toxins produced by the bacterium Clostridium botulinum. There are seven serologically distinct types of botulinum neurotoxins and they act near the neuromuscular junction, producing a flaccid paralysis. These neurotoxins are synthesized as single polypeptide chains which may be cleaved artificially or by endogenous proteases to more active dichain forms which are covalently held by disulfide linkage. The carboxy terminal 2/3 of the molecule is designated the heavy (H) chain, the rest is referred to as the light (L) chain. The neurotoxins have a molecular weight of approximately 150kD (for general review, Simpson, 1986)Simpson (1986) has proposed, by analogy to other bacterial toxins and viral infection models that the mode of action of these toxins proceed in three stages: binding to the surface of a cell, internalization and an enzymatic action. These functions can be separated biochemically; for example, the chains bind to the the cell surface and internalize. The enzymatic activity probably involves the L chain. This enzymatic poisoning step in neural cells is not known.

scholarly journals Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 888
Author(s):  
Hiro Uemachi ◽  
Yuuya Kasahara ◽  
Keisuke Tanaka ◽  
Takumi Okuda ◽  
Yoshihiro Yoneda ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Surface Protein ◽  
Functional Screening ◽  
Nucleic Acid Aptamers ◽  
Hybrid Type ◽  
Cell Internalization ◽  
Promising Target ◽  
A Cell ◽  
Exponential Enrichment

Nucleic acid aptamers have attracted considerable attention as next-generation pharmaceutical agents and delivery vehicles for small molecule drugs and therapeutic oligonucleotides. Chemical modification is an effective approach for improving the functionality of aptamers. However, the process of selecting appropriately modified aptamers is laborious because of many possible modification patterns. Here, we describe a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) approach for the generation of the artificial nucleic acid aptamers effective against human TROP2, a cell surface protein identified by drug discovery as a promising target for cancer therapy. Capillary electrophoresis SELEX was used for the pre-screening of multiple modified nucleic acid libraries and enrichment of TROP2 binding aptamers in the first step, followed by functional screening using cell-SELEX in the second step for the generation of cell-internalizing aptamers. One representative aptamer, Tac-B1, had a nanomolar-level affinity to human TROP2 and exhibited elevated capacity for internalization by cells. Because of the growing interest in the application of aptamers for drug delivery, our hybrid selection approach has great potential for the generation of functional artificial nucleic acid aptamers with ideal modification patterns in vitro.

N-Terminal amino acid sequence of rat tonin: homology with serine proteases

1978 ◽  
Vol 56 (9) ◽  
pp. 920-925 ◽  
Author(s):  
N. G. Seidah ◽  
R. Routhier ◽  
M. Caron ◽  
M. Chrétien ◽  
S. Demassieux ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Terminal Sequence ◽  
Renin Substrate ◽  
Amino Terminal ◽  
Single Polypeptide Chain ◽  
Serine Protease Family ◽  
Terminal Amino ◽  
Single Polypeptide ◽  
Carboxy Terminal ◽  
Amino Terminal Sequence

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residue s of the single polypeptide chain composed of 272 amino acids. The se results showed an extensive homology with the sequence of many serine proteases of the trypsin–chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.

scholarly journals A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
Author(s):  
K Fischman ◽  
J C Edman ◽  
G M Shackleford ◽  
J A Turner ◽  
W J Rutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Translation System ◽  
Appearance Time ◽  
Acid Protein ◽  
Mouse Tissues ◽  
Free Translation ◽  
Alternatively Spliced ◽  
A Cell ◽  
Testis Cdna ◽  
Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

Partial characterization of an abnormal lactate dehydrogenase isoenzyme, LDH-1ex, in serum from a patient with hepatocellular carcinoma.

1989 ◽  
Vol 35 (5) ◽  
pp. 844-848
Author(s):  
D L Kalpaxis ◽  
E E Giannoulaki
Keyword(s):  
Hepatocellular Carcinoma ◽  
Lactate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Heat Stable ◽  
Single Polypeptide ◽  
Polypeptide Chains

Abstract Serum from a patient with hepatocellular carcinoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), LDH-1ex, that on electrophoresis on 10-g/L agarose gel migrated anodally to the LDH-1 band. This isoenzyme was partly purified by ultrafiltration and preparative electrophoresis. Gel chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies of the resulting LDH-1ex preparation suggested that this isoenzyme is probably a tetramer made up of four single polypeptide chains (monomers), each having a molecular mass of about 32,000 Da. LDH-1ex was heat stable and reacted more readily with 2-hydroxybutyrate than did the slower migrating LDH-4 and LDH-5 isoenzymes. LDH-1ex showed no activity when lactate was omitted from the substrate solution or replaced by ethanol.

scholarly journals Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

2006 ◽  
Vol 74 (10) ◽  
pp. 5617-5624 ◽  
Author(s):  
Fang Cai ◽  
Carrie B. Adrion ◽  
James E. Keller
Keyword(s):  
Culture Medium ◽  
Reaction Model ◽  
Cultured Neurons ◽  
Total Inhibition ◽  
Botulinum Neurotoxins ◽  
A Cell ◽  
Proteolytic Activities ◽  
Living Neurons ◽  
Subsequent Addition

ABSTRACT Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

scholarly journals Dual binding motifs underpin the hierarchical association of perilipins1–3 with lipid droplets

2019 ◽  
Vol 30 (5) ◽  
pp. 703-716 ◽  
Author(s):  
Dalila Ajjaji ◽  
Kalthoum Ben M'barek ◽  
Michael L. Mimmack ◽  
Cheryl England ◽  
Haya Herscovitz ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Tissue Type ◽  
Binding Motifs ◽  
Amino Terminal ◽  
A Cell ◽  
Oil Water ◽  
Carboxy Terminal ◽  
Hierarchical Manner

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a “hierarchical” manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1–3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both “naked” and phospholipid-coated oil–water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1–3 to LDs.

scholarly journals Biosynthesis of pro-C3, a precursor of the third component of complement.

1977 ◽  
Vol 146 (3) ◽  
pp. 759-765 ◽  
Author(s):  
V Brade ◽  
R E Hall ◽  
H R Colten
Keyword(s):  
Disulfide Bonds ◽  
Polypeptide Chain ◽  
Peritoneal Macrophages ◽  
Tissue Cultures ◽  
The Third ◽  
Single Polypeptide Chain ◽  
Single Polypeptide ◽  
Polypeptide Chains ◽  
Third Component Of Complement

A precusor of the third component of complement, pro-C3, was detected in studies of cell-free synthesis and intracellularly in homogenates of liver tissue cultures. The molecular weight of pro-C3 was indistinguishable from that of intact native C3 secreted in vitro by liver or peritoneal macrophages, but its structure was different. Pro-C3 is a single polypeptide chain, whereas C3 secreted by cells in culture consists of two polypeptide chains (mol wt 120,000 and 76,000) linked by disulfide bonds.

The structure of some proteins as revealed by an x-ray scattering method

1955 ◽  
Vol 247 (932) ◽  
pp. 409-439 ◽  
Keyword(s):  
Protein Molecule ◽  
Helical Chain ◽  
Scattering Method ◽  
X Rays ◽  
Single Polypeptide Chain ◽  
X Ray Scattering ◽  
Synthetic Polypeptides ◽  
Single Polypeptide ◽  
Polypeptide Chains ◽  
Parallel Fashion

The intensity of scattering of monochromatic X-rays by isotropic specimens of thirty proteins (or protein fractions) and two synthetic polypeptides has been measured by means of a proportionalcounter diffractometer. The curves fall into only three essentially different types, calleda, /? and y. The a-proteins are the most numerous and some twenty have been examined; all the intensity curves are very similar and suggest that a single polypeptide-chain configuration is common to all as the predominant constituent. This predominant configuration is considered to be best represented by one of the variants of the a-helix of Pauling & Corey (1951 a ) and Pauling, Corey & Branson (1951) to which the term aj-helix is given. The method of interpretation used was to calculate the intensity of scattering that would be produced by independent polypeptide chains in random orientation, various regular configurations being assigned to them in accordance with existing hypotheses. In this way, the scattering characteristics of the following helical chain configurations were derived: a, y, 7r, 4 13 , 3 10 , 3 8 and 2 7 ; in the first five mentioned, the two alternative positions of the ^-carbon atom were considered. Comparison with the observed curves for a -proteins showed that the curve for theaj-helix was in best agreement with experiment, and with the synthetic polypeptides the correlation was particularly convincing. It was further shown that the several helices, or parts of helices, that constitute a whole protein molecule are usually packed together compactly in a nearly parallel fashion; in certain cases, a more pronounced skewness is indicated.

scholarly journals Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells.

1988 ◽  
Vol 106 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cell Types ◽  
Beta Tubulin ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
A Cell ◽  
Complex Regulation ◽  
Carboxy Terminal ◽  
Different Cell Types

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families.

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