scholarly journals Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽  
2021 ◽  
Author(s):  
Osheiza Abdulmalik ◽  
Noureldien H. E. Darwish ◽  
Vandhana Muralidharan-Chari ◽  
Maii Abu Taleb ◽  
Shaker A. Mousa
Keyword(s):  
Sickle Cell ◽  
Inflammatory Responses ◽  
Anticoagulant Activity ◽  
Single Point ◽  
Bleeding Complications ◽  
Single Point Mutation ◽  
Repeated Dosing ◽  
Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

scholarly journals Sickling Cells, Cyclic Nucleotides, and Protein Kinases: The Pathophysiology of Urogenital Disorders in Sickle Cell Anemia

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Mário Angelo Claudino ◽  
Kleber Yotsumoto Fertrin
Keyword(s):  
Protein Kinases ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Cyclic Nucleotides ◽  
Single Point ◽  
Clinical Manifestations ◽  
Single Point Mutation ◽  
Tract Infections

Sickle cell anemia is one of the best studied inherited diseases, and despite being caused by a single point mutation in theHBBgene, multiple pleiotropic effects of the abnormal hemoglobin S production range from vaso-occlusive crisis, stroke, and pulmonary hypertension to osteonecrosis and leg ulcers. Urogenital function is not spared, and although priapism is most frequently remembered, other related clinical manifestations have been described, such as nocturia, enuresis, increased frequence of lower urinary tract infections, urinary incontinence, hypogonadism, and testicular infarction. Studies on sickle cell vaso-occlusion and priapism using bothin vitroandin vivomodels have shed light on the pathogenesis of some of these events. The authors review what is known about the deleterious effects of sickling on the genitourinary tract and how the role of cyclic nucleotides signaling and protein kinases may help understand the pathophysiology underlying these manifestations and develop novel therapies in the setting of urogenital disorders in sickle cell disease.

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

scholarly journals The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

2000 ◽  
Vol 74 (23) ◽  
pp. 11027-11039 ◽  
Author(s):  
Eran Bacharach ◽  
Jason Gonsky ◽  
Kimona Alin ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Leukemia Virus ◽  
Single Point ◽  
Host Protein ◽  
Single Point Mutation ◽  
Gag Protein ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Carboxy Terminal

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

scholarly journals Robotic QM/MM-driven maturation of antibody combining sites

2016 ◽  
Vol 2 (10) ◽  
pp. e1501695 ◽  
Author(s):  
Ivan V. Smirnov ◽  
Andrey V. Golovin ◽  
Spyros D. Chatziefthimiou ◽  
Anastasiya V. Stepanova ◽  
Yingjie Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
In Vitro Selection ◽  
Single Point ◽  
Combinatorial Libraries ◽  
Single Point Mutation ◽  
Wild Type ◽  
Selection Of ◽  
Maturation Function

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

scholarly journals Cooperative Binding of Heat Shock Factor to the Yeast HSP82 Promoter In Vivo and In Vitro

1999 ◽  
Vol 19 (3) ◽  
pp. 1627-1639 ◽  
Author(s):  
Alexander M. Erkine ◽  
Serena F. Magrogan ◽  
Edward A. Sekinger ◽  
David S. Gross
Keyword(s):  
Heat Shock ◽  
Single Point ◽  
Heat Shock Factor ◽  
Single Point Mutation ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Before And After ◽  
Order Of Magnitude

ABSTRACT Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeastHSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced ∼100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.

Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 2054-2060 ◽  
Author(s):  
Elsa P. Bianchini ◽  
Judicael Fazavana ◽  
Veronique Picard ◽  
Delphine Borgel
Keyword(s):  
Anticoagulant Activity ◽  
Fold Increase ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Heparin Derivatives ◽  
Dose Dependent ◽  
Heparin Derivative ◽  
In Vivo Administration

Abstract Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.

Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain

1990 ◽  
Vol 10 (6) ◽  
pp. 2855-2862
Author(s):  
M C O'Brien ◽  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Kinase Activity ◽  
Single Point ◽  
Point Mutations ◽  
Soft Agar ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Single Point Mutation ◽  
Src Gene

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.

scholarly journals Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

1990 ◽  
Vol 10 (6) ◽  
pp. 2855-2862 ◽  
Author(s):  
M C O'Brien ◽  
Y Fukui ◽  
H Hanafusa
Keyword(s):  
Kinase Activity ◽  
Single Point ◽  
Point Mutations ◽  
Soft Agar ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Single Point Mutation ◽  
Src Gene

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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