Spectroscopy and Kinetics of Wild-Type and Mutant Tyrosine Hydroxylase: Mechanistic Insight into O2Activation

Degradation of recalcitrant phenolic syntan by electro-oxidation was investigated. The kinetics of mineralization of phenolic syntan was followed both in terms of TOC and COD measurements.

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Renitence vacuoles facilitate protection against phagolysosomal damage in activated macrophages

Molecular Biology of the Cell ◽

10.1091/mbc.e17-07-0486 ◽

2018 ◽

Vol 29 (5) ◽

pp. 657-668 ◽

Cited By ~ 3

Author(s):

Amanda O. Wong ◽

Matangi Marthi ◽

Zachary I. Mendel ◽

Brian Gregorka ◽

Michele S. Swanson ◽

...

Keyword(s):

Membrane Damage ◽

Morphological Features ◽

Activated Macrophages ◽

Membrane Ruffling ◽

Silica Bead ◽

Mechanistic Insight ◽

Silica Beads ◽

Lps Activation ◽

Kinetics Of ◽

Insight Into

As professional phagocytes, macrophages are susceptible to endolysosomal membrane damage inflicted by the pathogens and noxious particles they ingest. Whether macrophages have mechanisms for limiting such damage is not well understood. Previously, we reported a phenomenon, termed “inducible renitence,” in which lipopolysaccharide (LPS) activation of macrophages protected their endolysosomes against damage initiated by the phagocytosis of silica beads. To gain mechanistic insight into the process, we analyzed the kinetics of renitence and morphological features of LPS-activated versus resting macrophages following silica bead–mediated injury. We discovered novel vacuolar structures that form in LPS-activated but not resting macrophages following silica bead phagocytosis. Because of their correlation with renitence and damage-resistant nature, we termed these structures “renitence vacuoles” (RVs). RVs formed coincident with silica bead uptake in a process associated with membrane ruffling and macropinocytosis. However, unlike normal macropinosomes (MPs), which shrink within 20 min of formation, RVs persisted around bead-containing phagosomes. RVs fused with lysosomes, whereas associated phagosomes typically did not. These findings are consistent with a model in which RVs, as persistent MPs, prevent fusion between damaged phagosomes and intact lysosomes and thereby preserve endolysosomal integrity.

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Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

10.1101/2021.12.17.473246 ◽

2021 ◽

Author(s):

Sophie Hertel ◽

Richard Spinney ◽

Stephanie Xu ◽

Thomas E Ouldridge ◽

Richard Morris ◽

...

Keyword(s):

Secondary Structure ◽

Dna Sequences ◽

Physical Factors ◽

Biological Processes ◽

Physical Mechanisms ◽

Mechanistic Insight ◽

Sequence Dependent ◽

Dna Hybridisation ◽

Kinetics Of ◽

Insight Into

The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.

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Mechanistic Insight on Ras Inhibition Strategies in Cancer Therapy

Pharmaceutical Sciences ◽

10.34172/ps.2021.25 ◽

2021 ◽

Author(s):

Jeiran Pashapour-Anousheh ◽

Abolfazl Barzegar ◽

Mohammad Saeid Hejazi ◽

Nikoo Hasheminejad ◽

Hamid Reza Heidari ◽

...

Keyword(s):

Cancer Therapy ◽

Mechanism Of Action ◽

Cancer Type ◽

Ras Proteins ◽

Wild Type ◽

Drug Candidates ◽

Mechanistic Insight ◽

Ras Inhibition ◽

Specific Cancer ◽

Insight Into

Ras proteins are considered as one of the most critical cancer initiators. Mutations of this protein family lead to the continuous activation of the proliferation pathways. Therefore, many efforts have been taken to design the anti-mutant Ras drug candidates. Regardless of the development of promising inhibitors of Ras G12C mutant in a specific cancer type, there is no approved inhibitor of Ras mutants in the clinic. One of the significant limitations is to inhibit particular mutants and not to affect the wild-type Ras variants. Here we present a review on the mechanism of action of the Ras proteins to get a better insight into the strategies utilized to inhibit Ras-mutated cancers. The direct Ras inhibition strategies are then highlighted to obtain a better perspective of possible promising approaches to target Ras proteins in cancer therapy.

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CD19 Targeting of Lymphoid Malignancies by Novel Fc-Domain Engineered Monoclonal Antibody.

Blood ◽

10.1182/blood.v114.22.3725.3725 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3725-3725

Author(s):

Farrukh Awan ◽

Rosa Lapalombella ◽

Rossana Trotta ◽

Jonathan P Butchar ◽

Bo Yu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Nk Cells ◽

Nk Cell ◽

Wild Type ◽

Color Flow ◽

Mechanistic Insight ◽

Insight Into

Abstract Abstract 3725 Poster Board III-661 CD19 is a lineage-specific B-cell antigen, expressed at a high density on CLL cells, that contributes to B-cell receptor signaling but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified Fc-domain designed to enhance binding of FcγRIIIa that is predominately expressed on Natural Killer (NK)-cells. Utilizing freshly isolated chronic lymphocytic leukemia (CLL) patient B-cells we demonstrate that XmAb5574 lacks significant internalization seen with other anti-CD19 antibodies [maximum internalization for XmAb5574 was only 27.9% at 30-minutes (95%CI 14.5%, 41.4%)], thereby enhancing its ability to induce potent antibody-dependent cellular cytotoxicity (ADCC). Annexin V/PI flow cytometry analysis revealed that XmAb5574 mediates modest direct cytotoxicity not significantly different from Rituximab (0.6% increase, 95%CI -10.5%, 11.7%, p=0.91), and no complement mediated cytotoxicity (CDC) against primary CLL B-cells. Multi-color flow cytometry and monocyte derived macrophages (MDM) were used to assess XmAb5574 antibody dependent cellular phagocytosis (ADCP) against CLL cells and revealed no significant impact of the Fc-domain modification on MDM induced ADCP against CLL cells as compared to the wild type parental anti-CD19 antibody (12.37% vs. 10.51%, p=0.58). Interestingly, utilizing NK-cells and CLL cells isolated from normal donors and CLL patients, and employing autologous and allogeneic effector-target (E:T) conditions, XmAb5574 was found to mediate significantly higher ADCC when compared to the control humanized anti-CD19 non-engineered antibody (26.9% higher at E:T 25:1, p=0.0004 for allogeneic conditions, and 23.6% higher, p=0.004 for autologous conditions). ADCC mediated by XmAb5574 was also significantly higher as compared to Rituximab (33.5% higher at E:T 25:1, p<0.0001 for allogeneic conditions and 27.1% higher, p=0.004 for autologous conditions), a therapeutic antibody widely utilized in the treatment of CLL, hence confirming the functional in vitro efficacy and utility of the Fc-domain modification. By using inhibitor studies we further provide mechanistic insight into the XmAb5574–dependent ADCC mediated by NK-cells through a Granzyme B dependent mechanism. XmAb5574 also enhanced NK-cell activation as exhibited by an increased phosphorylation of Erk1/2 downstream of Fcγ receptor. The enhancement of subsequent cytolytic and secretory function was shown by the measurement CD107a up regulation on the surface of NK-cells (19.4% increase, p=0.005, as compared to wild type anti-CD19 antibody), and interferon-gamma release as measured by ELISA assays (6.4 times higher, p=0.007, as compared to wild type anti-CD19 antibody). Notably, enhanced NK-cell mediated ADCC observed with XmAb5574 against primary CLL B-cells could be augmented further by treatment with Lenalidomide (17.9% higher, p=0.04). These findings provide strong pre-clinical evidence for further clinical development of XmAb5574, both as monotherapy and in combination with Lenalidomide, for the therapy of CLL and related CD19+ B-cell malignancies. We also provide mechanistic insight into the utility and feasibility of Fc-domain engineering of specific antibodies, which will enhance their efficacy through an increased ability to recruit the innate immune system to more effectively control tumor progression. Disclosures: Desjarlais: Xencor: Employment.

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Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations

Biochemical Journal ◽

10.1042/bj20101672 ◽

2011 ◽

Vol 436 (1) ◽

pp. 113-121 ◽

Cited By ~ 12

Author(s):

Pernilla von Nandelstadh ◽

Rabah Soliymani ◽

Marc Baumann ◽

Olli Carpen

Keyword(s):

Morphological Changes ◽

Protein Accumulation ◽

Cleavage Sites ◽

Wild Type ◽

Molecular Defects ◽

Mechanistic Insight ◽

First Time ◽

Insight Into ◽

Biological Differences

MFM (myofibrillar myopathies) are caused by mutations in several sarcomeric components, including the Z-disc protein myotilin. The morphological changes typical of MFM include Z-disc alterations and aggregation of dense filamentous sarcomeric material. The causes and mechanisms of protein aggregation in myotilinopathies and other forms of MFM remain unknown, although impaired degradation may explain, in part, the abnormal protein accumulation. In the present paper we have studied the mechanisms regulating myotilin turnover, analysed the consequences of defective myotilin degradation and tested whether disease-causing myotilin mutations result in altered protein turnover. The results indicate that myotilin is a substrate for the Ca2+-dependent protease calpain and identify two calpain cleavage sites in myotilin by MS. We further show that myotilin is degraded by the proteasome system in transfected COS7 cells and in myotubes, and that disease-causing myotilinopathy mutations result in reduced degradation. Finally, we show that proteolysis-inhibitor-induced reduction in myotilin turnover results in formation of intracellular myotilin and actin-containing aggregates, which resemble those seen in diseased muscle cells. These findings identify for the first time biological differences between wt (wild-type) and mutant myotilin. The present study provides novel information on the pathways controlling myotilin turnover and on the molecular defects associated with MFM.

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