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T Loukianova

Publications and source records attributed to T Loukianova.

9 recordsLinked to original sources
PubMed1999

SERCA1a can functionally substitute for SERCA2a in the heart.

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an approximately 2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889-897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased ( approximately 1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

Adenosine Triphosphate↗
PubMed1998

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

Animals↗
PubMed1998

Variation in the proregion structure of heparin-binding EGF-like growth factor precursors.

In a previous study, we have isolated and characterized cDNA encoding a novel 'short form' of heparin-binding EGF-like growth factor (SF HB-EGF) (Loukianov et al., 1997). In the present work, we have found that cDNA for SF HB-EGF and for full-length HB-EGF are each represented by two variants, which we refer to as L and P forms. The L form is the previously known form of HB-EGF cDNA and encodes a leucine in position 33. The P form described in this report, encodes a proline in codon 33. The L33P substitution is predicted to cause a significant alteration in the proregion structure of SF HB-EGF and HB-EGF.

Amino Acid Sequence↗
PubMed1997

Expression of mRNA for a short form of heparin-binding EGF-like growth factor.

In this paper we report the cloning and characterization of cDNA encoding a novel, short form of heparin-binding EGF-like growth factor (SF HB-EGF), and show expression of specific mRNA in various tissues and cell types. Our data suggest that SF HB-EGF mRNA is a product of alternative splicing. Like normal HB-EGF, SF HB-EGF contains the signal peptide, the propeptide, the heparin-binding domain and the first two conservative disulfide loops of the EGF unit. Instead of the third disulfide loop, the spacer, the transmembrane and the cytoplasmic domains, SF HB-EGF has a nine amino acid tail.

Amino Acid Sequence↗
PubMed1997

Myosin heavy chain isoforms in smooth muscle.

In recent years, significant progress has been made toward understanding smooth muscle myosin heavy chain (SMHC) structure. Molecular cloning analysis has identified four different MHC isoforms. They are products of a single gene and result from alternative mRNA splicing. In addition, two non-muscle MHC isoforms are also expressed in smooth muscle cells. Studies show that SMHC expression is highly tissue specific and does not appear in cardiac or skeletal muscle cells. Each smooth muscle tissue is characterized by a specific pattern of MHC isoform expression that changes during development and disease. This review essentially focuses on SMHC isoforms and their expression in mammals.

Alternative Splicing↗
PubMed1997

Cloning DNA fragments between two adjacent/overlapping restriction sites using a "positive stuffer".

Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site. The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin. This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.

Binding Sites↗
PubMed1997

Efficient cloning method that selects the recombinant clones.

Directional cloning using cohesive ends is the most efficient cloning method. However, sometimes it is necessary to use blunt ends to clone a DNA fragment into the plasmid vector. Compared with that of cohesive ends, efficiency of blunt-end ligation is low. Compared with the native blunt ends (e.g., SmaI or EcoRV), blunt-end ligation is particularly difficult when blunt ends are derived from overhangs. This results in low efficiency of insertion and high background from self-ligation of the vector. To remedy the problem, we developed a "positive selector" cloning strategy that provides positive selection for the recombinant clones. It is particularly useful when making complex recombinant constructs and the choice of restriction sites is limited.

Cloning, Molecular↗
PubMed1994

Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K.

In human cells at least 20 different proteins or groups of proteins have been identified that are associated with hnRNAs. These proteins (designated A1-U) are highly abundant in the nucleus. In this study, we present the sequence of a novel cDNA clone, sub2.3, isolated from a human lymphocyte cDNA library. The predicted amino acid sequence shows homology to repeated domains in the human hnRNA binding protein K (hnRNP K), which are believed to be of functional importance. hnRNP K is among the major oligo(rC/dC) binding proteins in vertebrate cells and we show here that the protein product of sub2.3 also binds to oligo(dC). This is shown by a novel approach where we demonstrated specific binding of in vitro translated sub2.3 protein to biotinylated oligo(dC) which was immobilized on magnetic streptavidin-coated Dynabeads. Moreover we found that the sub2.3 transcript is expressed in a tissue dependent manner with the highest expression observed in several lymphoid tissues and skeletal muscle. The gene was also abundantly expressed in several lymphoid cell lines and the hepatoma cell line HepG2 while a low expression was observed in the HL60 myeloid cell line and in the HeLa cervical carcinoma cell line. In conclusion, this study presents the cDNA sequence of a novel transcript which shows tissue specific expression and encodes a protein with oligo(dC) binding specificity in vitro.

Amino Acid Sequence↗