Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death.

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.

Protein kinase C activation increases release of secreted amyloid precursor protein without decreasing Abeta production in human primary neuron cultures.

Overexpression and altered metabolism of amyloid precursor protein (APP) resulting in increased 4 kDa amyloid beta peptide (Abeta) production are believed to play a major role in Alzheimer's disease (AD). Therefore, reducing Abeta production in the brain is a possible therapy for AD. Because AD pathology is fairly restricted to the CNS of humans, we have established human cerebral primary neuron cultures to investigate the metabolism of APP. In many cell lines and rodent primary neuron cultures, phorbol ester activation of protein kinase C (PKC) increases the release of the secreted large N-terminal fragment of amyloid precursor protein (sAPP) and decreases Abeta release (; ; ). In contrast, we find that PKC activation in human primary neurons increases the rate of sAPP release and the production of APP C-terminal fragments and 4 kDa Abeta. Our results indicate species- and cell type-specific regulation of APP metabolism. Therefore, our results curtail the use of PKC activators in controlling human brain Abeta levels.

New adenovirus vectors for protein production and gene transfer.

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10-15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression.

Amyloid beta peptide of Alzheimer's disease downregulates Bcl-2 and upregulates bax expression in human neurons.

Neuronal apoptosis is a suspected cause of neurodegeneration in Alzheimer's disease (AD). Increased levels of amyloid beta peptide (Abeta) induce neuronal apoptosis in vitro and in vivo. The underlying molecular mechanism of Abeta neurotoxicity is not clear. The normal concentration of Abeta in cerebrospinal fluid is 4 nM. We treated human neuron primary cultures with 100 nM amyloid beta peptides Abeta(1-40) and Abeta(1-42) and the control reverse peptide Abeta(40-1). We find that although little neuronal apoptosis is induced by either peptide after 3 d of treatment, Abeta(1-42) provokes a rapid and sustained downregulation of a key anti-apoptotic protein, bcl-2, whereas it increases levels of bax, a protein known to promote cell death. In contrast, the Abeta(1-40) downregulation of bcl-2 is gradual, although the levels are equivalent to those of Abeta(1-42)-treated neurons by 72 hr of treatment. Abeta(1-40) does not upregulate bax levels. The control, reverse peptide Abeta(40-1), does not affect either bcl-2 or bax protein levels. In addition, we found that the Abeta(1-40)- and Abeta(1-42)- but not Abeta(40-1)-treated neurons had increased vulnerability to low levels of oxidative stress. Therefore, we propose that although high physiological amounts of Abeta are not sufficient to induce apoptosis, Abeta depletes the neurons of one of its anti-apoptotic mechanisms. We hypothesize that increased Abeta in individuals renders the neurons vulnerable to age-dependent stress and neurodegeneration.

Overexpression of vsr in Escherichia coli is mutagenic.

Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.

Targeting glucose oxidase at aspartate and glutamate residues with organic two-electron redox mediators.

The bimolecular rate constants for the reactions of five organic two-electron redox mediators with reduced glucose oxidase (GOx) were determined by measuring voltammetric electrocatalytic currents at glassy carbon electrodes in the presence of excess glucose under anaerobic conditions. The mediators studied were thionine, brilliant cresyl blue, azure A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M-1 s-1) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10(6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate residues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dopamine groups were obtained, with activities of 50% and 75%, respectively, relative to native GOx in a dye-peroxidase assay. Although the daunomycin derivative did not show measurable intramolecular electron-transfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH2 groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, the currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s-1) could be determined. This value is > 20 times larger than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling reagents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.