Search

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 μM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

Phosgene is classified as a chemical warfare agent, yet data on its short-duration high concentration toxicity in a nose-only exposure rat model is sparse and inconsistent. Hence, an exposure system for short-term/high concentration exposure was developed and characterized. Herein, we report the median lethal concentration (LC50) for a 10-min nasal exposure of phosgene in a 24-h rat survival model. Male Wistar rats (Envigo) weighing 180-210 g on the day of exposure, were exposed to phosgene gas via nose-only inhalation using a system specifically designed to allow the simultaneous exposure and quantification of phosgene. After 24 h, the surviving rats were euthanized, the lung/body mass ratio determined, and lung tissues analyzed for histopathology. Increased terminal airway edema in the lungs located primarily at the alveoli (resulting in an increased lung/body mass ratio) coincided with the observed mortality. An LC50 value of 129.2 mg/m3 for a 10-min exposure was determined. Furthermore, in agreement with other highly toxic compounds, this study reveals a LC50 concentration value supportive of a nonlinear toxic load model, where the toxic load exponent is >1 (ne = 1.17). Thus, in line with other chemical warfare agents, phosgene toxicity is predicted to be more severe with short-duration, high-concentration exposures than long-duration, low-concentration exposures. This model is anticipated to be refined and developed to screen novel therapeutics against relevant short-term high concentration phosgene exposures expected from a terrorist attack, battlefield deployment, or industrial accident.

BackgroundAlthough early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress.ObjectivesThis study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes.MethodsA left coronary artery occlusion (40 min) reperfusion (3 h) model was used in 31 rabbits. Of these, 12 rabbits received the fusion protein (Fv-HSP72) intravenously. The remaining 19 control rabbits received a molar equivalent of 3E10Fv alone (n = 6), HSP72 alone (n = 6), or phosphate-buffered saline (n = 7). Serial echocardiographic examinations were performed to assess left ventricular function before and after reperfusion. Micro-single-photon emission computed tomography imaging of 99mTc-labeled annexin-V was performed with micro-computed tomography scanning to characterize apoptotic damage in vivo, followed by gamma counting of the excised myocardial specimens to quantify cell death. Histopathological characterization of the myocardial tissue and sequential cardiac troponin I measurements were also undertaken.ResultsMyocardial annexin-V uptake was 43% lower in the area at risk (p = 0.0003) in Fv-HSP72-treated rabbits compared with control animals receiving HSP72 or 3E10Fv alone. During reperfusion, troponin I release was 42% lower and the echocardiographic left ventricular ejection fraction 27% higher in the Fv-HSP72-treated group compared with control animals. Histopathological analyses confirmed penetration of 3E10Fv-containing molecules into cardiomyocytes in vivo, and treatment with Fv-HSP72 showed fewer apoptotic nuclei compared with control rabbits.ConclusionsSingle-dose administration of Fv-HSP72 fusion protein at the time of reperfusion reduced myocardial apoptosis by almost one-half and improved left ventricular functional recovery after myocardial ischemia-reperfusion injury in rabbits. It might have potential to serve as an adjunct to early reperfusion in the management of myocardial infarction.

Cancer cells with defects in DNA repair are highly susceptible to DNA-damaging agents, but delivery of therapeutic agents into cell nuclei can be challenging. A subset of lupus autoantibodies is associated with nucleolytic activity, and some of these antibodies are capable of nuclear penetration. We hypothesized that such antibodies might have potential as therapeutic agents targeted towards DNA repair-deficient malignancies. We identified the lupus autoantibody 5C6 as a cell-penetrating nucleolytic antibody and found that 5C6 has a differential effect on a matched pair of BRCA2-proficient and deficient DLD1 colon cancer cells. 5C6 selectively induced γH2AX in, and suppressed the growth of, the BRCA2-deficient cells. These findings demonstrate the potential utility of 5C6 in targeted therapy for DNA repair-deficient malignancies and strengthen the rationale for studies of additional lupus autoantibodies in order to identify the best candidates for development as therapeutic agents. In addition, the toxic effect of 5C6 on BRCA2-deficient cells provides further support for the hypothesis that some lupus autoantibodies contribute to the lower risk of specific cancers associated with systemic lupus erythematosus.

Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cells in vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.

Defects in dendritic spines are common to several forms of cognitive deficits, including mental retardation and Alzheimer disease. Because mutation of p21-activated kinase (PAK) can lead to mental retardation and because PAK-cofilin signaling is critical in dendritic spine morphogenesis and actin dynamics, we hypothesized that the PAK pathway is involved in synaptic and cognitive deficits in Alzheimer disease. Here, we show that PAK and its activity are markedly reduced in Alzheimer disease and that this is accompanied by reduced and redistributed phosphoPAK, prominent cofilin pathology and downstream loss of the spine actin-regulatory protein drebrin, which cofilin removes from actin. We found that β-amyloid (Aβ) was directly involved in PAK signaling deficits and drebrin loss in Aβ oligomer–treated hippocampal neurons and in the Appswe transgenic mouse model bearing a double mutation leading to higher Aβ production. In addition, pharmacological PAK inhibition in adult mice was sufficient to cause similar cofilin pathology, drebrin loss and memory impairment, consistent with a potential causal role of PAK defects in cognitive deficits in Alzheimer disease. [ABSTRACT FROM AUTHOR]