scholarly journals Changes in the surface membrane during myoblast fusion

1980 ◽  
Vol 190 (3) ◽  
pp. 647-652 ◽  
Author(s):  
D H Curtis ◽  
K J Micklem ◽  
K Gill ◽  
C A Pasternak
Keyword(s):  
Amino Acids ◽  
Chick Embryo ◽  
Surface Membrane ◽  
Surface Protein ◽  
Myoblast Fusion ◽  
The Other ◽  
Methyl Glucoside ◽  
Simple Diffusion ◽  
Approximate Doubling

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.

scholarly journals Nutritional availability of amino acids from protein cross-linked to protect against degradation in the rumen

1984 ◽  
Vol 52 (2) ◽  
pp. 239-247 ◽  
Author(s):  
John R. Ashes ◽  
Jim L. Mangan ◽  
Gurcharn S. Sidhu
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Medicago Sativa ◽  
Terminal Ileum ◽  
Milk Protein ◽  
The Other ◽  
Molar Basis ◽  
Lactating Goats ◽  
Total Milk

1. Casein was labelled with pairs of radioactive amino acids, lysine, tyrosine and leucine, one with I4C and the other with 3H, by jugular infusion into lactating goats followed by isolation of the double-labelled casein from the milk. Total milk protein was similarly labelled by jugular infusion of [35S]cystine. U-14C-labelled fraction- 1 leaf protein was isolated from lucerne (Medicago sativa) grown in an atmosphere of 14C022. The proteins were treated withdifferent levels(333 and667 mmol/kgprotein) offormaldehyde, glutaraldehyde and glyoxal.3. Absorption from the small intestine was measured in sheep with fistulas in the abomasum and terminal ileum, using Cr-EDTA as the digesta flow marker, by introducing radioactive casein into the abomasum.4. Lysine, tyrosine and cystine became increasingly unavailable for absorption from the small intestine of sheep with increasing levels of aldehyde. At the lower level (333 mmol/kg) the proportions of the amino acids that were unavailable were 0.192, 0.051 and 0.123 respectively. At the higher level of formaldehyde (667 mmol/kg) the corresponding values were 0.335, 0.201 and 0.432 respectively. Leucine was not made unavailable with formaldehyde.5. The proportions of lysine, tyrosine and leucine that were unavailable were higher, on a molar basis, after treatment of the proteins with the dialdehydes glutaraldehyde and glyoxal than after treatment with formaldehyde. However, the extent of protein protection provided by the dialdehydes in the rumen, measured using an in vitro procedure, was lower.

scholarly journals Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

2020 ◽  
Vol 477 (5) ◽  
pp. 905-923 ◽  
Author(s):  
Sunil Singh ◽  
J. Sivaraman
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Cancer Metastasis ◽  
E3 Ligase ◽  
The Other ◽  
Hect Domain ◽  
Loop Region ◽  
The Stability

The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

scholarly journals The sites of hydrolysis of dipeptides containing leucine and glycine by rat jejunum in vitro

1969 ◽  
Vol 114 (4) ◽  
pp. 855-861 ◽  
Author(s):  
E. B. Fern ◽  
R. C. Hider ◽  
D. R. London
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
The Other ◽  
Peptidase Activity ◽  
Rat Jejunum ◽  
Effective Inhibitor ◽  
Inhibitory Effects ◽  
Molar Basis ◽  
Hydrolysis Of

1. The effect of peptides containing leucine and glycine on accumulation of leucine and glycine by everted jejunal rings was studied. 2. It was shown that, on a molar basis, leucyl-leucine is a more effective inhibitor of uptake of [14C]leucine than is either leucylglycine or glycyl-leucine. These latter dipeptides behave alike. 3. The concentration of the dipeptides and their constituent amino acids in both the incubation medium and the tissue has been followed in these experiments by amino acid analysis. No leucine-containing peptides were observed in the tissue. 4. The inhibitory effects of the mixed dipeptides are altered by pH changes in an analogous way to the alterations in peptidase activity. 5. The experimental results indicate that leucine-containing peptides are hydrolysed before the transport step. 6. Glycylglycine, on the other hand, has only a small effect on the accumulation of glycine, although large amounts of the peptide accumulate unchanged in the tissue. This suggests that glycylglycine is taken up by a different mechanism to that for the leucine dipeptides.

scholarly journals The H5N1 Influenza Virus NS Genes Selected after 1998 Enhance Virus Replication in Mammalian Cells

2007 ◽  
Vol 81 (15) ◽  
pp. 8112-8121 ◽  
Author(s):  
Karen Y. Twu ◽  
Rei-Lin Kuo ◽  
Jesper Marklund ◽  
Robert M. Krug
Keyword(s):  
Amino Acids ◽  
Virus Replication ◽  
Influenza A ◽  
The Other ◽  
Intrinsic Defect ◽  
Human Influenza ◽  
Beta Interferon ◽  
H5n1 Influenza ◽  
Ns1a Protein

ABSTRACT The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.

Structure of the protein encoded by the chicken proto-oncogene c-myb

1986 ◽  
Vol 6 (11) ◽  
pp. 3677-3684
Author(s):  
S Gerondakis ◽  
J M Bishop
Keyword(s):  
Amino Acids ◽  
The Other ◽  
Biological Functions ◽  
Avian Myeloblastosis Virus ◽  
Systematic Exploration ◽  
Initiation Of Translation ◽  
Single Substitution

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.

Synthesis of globin chains in the erythropoietic sites of the early chick embryo

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 153-165
Author(s):  
L. Fucci ◽  
C. Cirotto ◽  
L. Tomei ◽  
G. Geraci
Keyword(s):  
Chick Embryo ◽  
Relative Concentration ◽  
The Other ◽  
Specific Mechanism ◽  
In Ovo ◽  
Globin Chains ◽  
Immunofluorescence Labelling ◽  
Α Chain

The synthesis of globins in the chick embryo before the onset of circulation has been studied in situ by specific immunofluorescence labelling of embryonic sections and by labelling newly synthesized proteins in ovo and in vitro in embryonic explants with [3H]leucine. The presence of major primitive haemoglobins is observed by 28 h of incubation. The minor primitive haemoglobins become detectable by immunofluorescence after 40 h of development, shortly before the onset of circulation. 3H-labelling shows that one definitive α chain is synthesized, though in low concentration, from the initial globin detection. The other definitive α chain is observed in embryos of at least 40 h of development. The relative concentration of the two definitive α chains changes rapidly with development indicating a specific mechanism of regulation. An erythropoietic site is observed in the wall of the dorsal aorta in embryos of about 45–50 h of development. From the initial detection, those cells contain all four primitive embryonic haemoglobins, in contrast to what is observed for the cells of the blood islands.

Uptake of a Series of Neutral Dipeptides Including l-Alanyl-l-Alanine, Glycylglycine and Glycylsarcosine by Hamster Jejunum in Vitro

1984 ◽  
Vol 67 (5) ◽  
pp. 541-549 ◽  
Author(s):  
D. M. Matthews ◽  
D. Burston
Keyword(s):  
Amino Acids ◽  
Free Amino ◽  
Inhibitory Effect ◽  
Progressive Increase ◽  
The Other ◽  
Uptake System ◽  
Apparent Affinity ◽  
Inhibitory Ability ◽  
Kinetics Of

1. This paper is the last of a set reporting an investigation of the kinetics of jejunal uptake and inhibitory ability of a series of neutral dipeptides, glycylglycine, l-ananyl-l-alanine, l-valyl-l-valine and l-leucyl-l-leucine, with progressively longer and more lipophilic side chains. 2. The results suggested that at pH 5, uptake of l-alanyl-l-alanine, like that of l-valyl-l-valine and l-leucyl-l-leucine, was the result of two processes, uptake of intact peptide and uptake of free amino acid released extracellularly. On the other hand, uptake of glycylglycine was entirely in the form of intact peptide. In contrast to uptake of l-valyl-l-valine and l-leucyl-l-leucine, the proportion of intact l-alanyl-l-alanine taken up by mediated transport was greatest at the lowest concentration studied and smallest at the highest concentration. 3. Taking the series of results as a whole, whereas the corresponding series of amino acids, glycine, l-alanine, l-valine and l-leucine, showed a progressive increase in apparent affinity for uptake and a decrease in Vmax., we could find no such regular progression with the peptides. 4. The results of work on inhibition of uptake of one dipeptide by another were unexpectedly complex. Examples were the very powerful inhibitory effect of l-valyl-l-valine on uptake of glycylsarcosine, not suggested by the Kt of the former peptide, and the failure of glycylsarcosine to cause complete inhibition of uptake of l-alanyl-l-alanine and l-leucyl-l-leucine, though it could completely inhibit uptake of l-valyl-l-valine. There may be more than one uptake system for intact peptides, but we cannot yet suggest an explanation for all the results on inhibitions of uptake.

HETEROKARYON ANALYSIS OF THE GENETIC CONTROL OF PIGMENT SYNTHESIS IN CHICK EMBRYO MELANOCYTES

Genetics ◽  
1982 ◽  
Vol 102 (3) ◽  
pp. 557-569
Author(s):  
L M Wilkins ◽  
J A Brumbaugh ◽  
J W Moore
Keyword(s):  
Chick Embryo ◽  
Genetic Control ◽  
Regulatory Elements ◽  
The Other ◽  
Wild Type ◽  
Mutant Type ◽  
Pigment Synthesis ◽  
Dominant Mutant

ABSTRACT The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, ey and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with 3H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-ey combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.

Influx of Two Dipeptides, Glycylsarcosine and l-glutamyl-l-glutamic Acid, into hamster Jejunum in Vitro

1979 ◽  
Vol 56 (1) ◽  
pp. 15-23 ◽  
Author(s):  
D. M. Matthews ◽  
R. H. Gandy ◽  
E. Taylor ◽  
D. Burston
Keyword(s):  
Glutamic Acid ◽  
Brush Border ◽  
Transport Mechanism ◽  
Inhibitory Effect ◽  
The Other ◽  
Peptide Transport ◽  
High Concentration ◽  
Simple Diffusion ◽  
Diffusion Component

1. This paper reports an investigation of whether the dipeptides glycylsarcosine and l-glutamyl-l-glutamic acid share a single mediated transport mechanism into hamster jejunum, or whether one of these peptides is transported in part by a transport mechanism unavailable to the other. It describes the kinetics of influx of glycylsarcosine and of l-glutamyl-l-glutamic acid into rings of everted hamster jejunum in vitro, incubations being carried out at pH 5 in order to minimize brush-border and intra-medium hydrolysis of l-glutamyl-l-glutamic acid, so that peptide transport rather than a mixture of peptide transport and transport of free glutamic acid was studied. With glycylsarcosine, brush-border and intra-medium hydrolysis are negligibly small. 2. Estimates of the simple diffusion component in transport of each peptide were made by treating each of the substrates as a competitive inhibitor of its own mediated transport (assuming that mediated transport conforms to simple Michaelis-Menten kinetics), extrapolating the observed inhibitory effect over a range of concentrations to an infinitely high concentration of inhibitor, and estimating the transport component remaining at such a concentration. This component in transport would be expected to represent transport by simple diffusion, and this assumption was supported by the observation that for glycylsarcosine the uninhibitable component in transport was linearly proportional to substrate concentration; with l-glutamyl-l-glutamic acid the observations were too few to provide this demonstration. Estimates of apparent Kt and Vmax. for mediated transport of both peptides are given. Before correction for simple diffusion, linearizing plots were clearly biphasic for both peptides; after correction for simple diffusion, they became linear, providing no evidence for transport of either peptide by more than one mediated transport system, though not excluding the possibility of multiple systems. 3. Measurement of influx of [14C]Gly-Sar over a range of concentrations both alone and in the presence of a constant concentration of Glu-Glu showed that after correction for the non-mediated component in influx of Gly-Sar (simple diffusion), influx of this peptide conformed to Michaelis-Menten kinetics and the inhibitory effect of Glu-Glu on influx of Gly-Sar appeared to be competitive. The extent of inhibition corresponded well with that predicted from the Kt values of the two peptides. 4. Measurement of influx of [14C]Gly-Sar (1 mmol/l) in the presence of a range of concentrations of Glu-Glu, with extrapolation of the inhibitory effect of Glu-Glu to an infinitely high concentration of this peptide, showed that at such a concentration mediated influx of Gly-Sar was completely abolished, influx being reduced to the simple diffusion component in total influx of [14C]Gly-Sar. Measurement of influx of [14C]Glu-Glu (1 mmol/l) in the presence of a range of concentrations of Gly-Sar, with extrapolation of the inhibitory effect of Gly-Sar to an infinitely high concentration of this peptide, showed that at such a concentration mediated influx of Glu-Glu was completely abolished, influx being reduced to the simple diffusion component in total influx of [14C]Glu-Glu. 5. The results are compatible with the conclusion that Gly-Sar and Glu-Glu are taken up by the absorptive cells by a single mediated mechanism. They do not exclude the possibility that these peptides are taken up by multiple common mechanisms, but they do appear to exclude the possibility that at the substrate concentration used (1 mmol/l) there is appreciable uptake of one of the peptides by a system unavailable to the other.

scholarly journals Functional Domains Present in the Mycobacterial Hemagglutinin, HBHA

1999 ◽  
Vol 181 (24) ◽  
pp. 7464-7469 ◽  
Author(s):  
Giovanni Delogu ◽  
Michael J. Brennan
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Surface Protein ◽  
Functional Domains ◽  
Heparin Binding ◽  
Coiled Coil Region ◽  
Host Tissues

ABSTRACT Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an α-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.

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