Juliann G. Kiang

Heat Shock Protein 70 kDa ☆: Molecular Biology, Biochemistry, and Physiology

Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic. In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults. Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer. Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases. Most of the HSPs interact with other proteins in cells and alter their function. These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions. In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways. The clinical importance and therapeutic potential of HSPs are discussed.

Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn2+ and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn2+-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.

Depressor and natriuretic activities of several atrial peptides

Mammalian atria contain peptides that have depressor and natriuretic activities. Four peptides, atriopeptin I to III (AP I to III) and alpha-human atrial natriuretic factor (alpha-hANP), were synthesized and assayed in the urethane-anesthetized rat for cardiovascular changes and natriuretic activities. All four peptides produced depressor responses and natriuresis. The relative activities were: alpha-hANP = AP III greater than AP II greater than AP I. The disappearance of iodinated AP III from plasma was rapid, with an estimated half-life of 2.5 min. Atriopeptin III was degraded by tissue homogenates, the relative activities being: kidney greater than liver greater than lung greater than plasma greater than heart. The HPLC profile of AP III suggested that smaller peptide fragments were formed after incubation with kidney homogenates. The degradation of AP III was inhibited by bestatin, an aminopeptidase inhibitor, and SQ 20881, a carboxypeptidase inhibitor.

Overexpression of HSP-70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity

This laboratory reported previously that overexpressed heat shock protein 70 kDa (HSP‐70) inhibited the activation of its transcriptional factor, HSF1. We had conducted experiments to understand the mechanisms whereby HSP‐70 down‐regulated the activation of HSF1. Genetically overexpressed HSP‐70 had no effects on the HSF1 level in cytosol, but significantly inhibited phosphorylation of HSF1 in the nucleus. Transfection of cells with HSF1 cDNA resulted in increases in the unphosphorylated, but not phosphorylated, HSF1 levels in both the cytosol and nucleus. Because serine phosphorylation of various proteins was reduced in HSP‐70 cDNA‐transfected cells, we measured the activity of enzymes involved in serine phosphorylation. Overexpressed HSP‐70 significantly inhibited the enzymatic activities of protein kinase A (PKA by 73 and 62% in the cytosol and membrane‐bound fraction, respectively) and protein kinase C (PKC by 61% in membrane‐bound fraction), whereas it activated that of protein phosphatase (PP by 33 and 86% in the cytosol and the membrane‐bound fraction, respectively). Forskolin (a PKA stimulator), PMA (a PKC stimulator), and okadaic acid (an inhibitor of PP) were used to investigate whether HSP‐70‐induced changes in PKA, PKC, and PP were responsible for the HSF1 dephosphorylation. For‐skolin did not change nuclear HSF1 phosphorylation, suggesting that decreases in PKA activity in HSP‐70 overexpressing cells is not associated with HSF1 phosphorylation. PMA and okadaic acid induced an increase in HSF1 phosphorylation in both vector‐and HSP‐70 cDNA‐transfected cells, although levels of phosphorylated HSF1 in HSP‐70 cDNA‐transfected cells were lower than those in vector‐transfected cells. The PMA‐induced increase in HSF1 phosphorylation in HSP‐70 cDNA‐transfected cells was blocked by pretreatment with stau‐rosporine, a PKC inhibitor. These results suggest that overexpression of HSP‐70 inhibits phosphorylation of HSF1 at serine residues by activating PP and inhibiting PKC activity.—Ding, X. Z., Tsokos, G. C., Kiang, J. G. Overexpression of HSP‐70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity FASEB J. 12, 451–459 (1998)

Subpicosecond time-resolved Raman studies of LO phonons in GaN: Dependence on photoexcited carrier density

Subpicosecond time-resolved Raman spectroscopy has been used to measure the lifetime of the LO phonon mode in GaN for photoexcited electron-hole pair density ranging from 1016to2×1019cm−3. The lifetime has been found to decrease from 2.5ps, at low density, to 0.35ps, at the highest density. The experimental findings should help resolve the recent controversy over the lifetime of LO phonon mode in GaN.

Wound Trauma Increases Radiation-Induced Mortality by Activation of iNOS Pathway and Elevation of Cytokine Concentrations and Bacterial Infection

Abstract Although it is documented that concurrent wounding increases mortality from radiation injury, the molecular mechanism of combined injury is unknown. In this study, mice were exposed to γ radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, reducing the LD50/30 from 9.65 Gy to 8.95 Gy. Analyses of histopathology, inducible nitric oxide synthase (iNOS), and serum cytokines were performed on mouse ileum and skin at various times after 9.75 Gy and/or wounding. In the ileum, the villi were significantly shortened 3 days postirradiation but not after wounding; combined injury resulted in decreased villus width and tunica muscularis thickness. The skin of mice subjected to combined injury was less cellular and had a smaller healing bud than the skin of mice subjected to wounding alone. Combined injury significantly delayed wound closure times; it also prolonged the increased levels of iNOS protein in the skin and ileum. iNOS up-regulation was correlated with increases in transcription factors, including NF-κB and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1β, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-γ, MCP-1, MIP-1α and MIP-1β. Combined injury resulted in early detection of bacteria in the blood of the heart and liver, whereas radiation alone resulted in later detection of bacteria; only a transient bacteremia occurred after wounding alone. Results suggest that enhancement of iNOS, cytokines and bacterial infection triggered by combined injury may contribute to mortality. Agents that inhibit these responses may prove to be therapeutic for combined injury and may reduce related mortality.

Inducible heat shock protein 70 kD and inducible nitric oxide synthase in hemorrhage/resuscitation-induced injury.

ABSTRACTInducible heat shock protein 70 kD (HSP-70i) has been shown to protect cells, tissues, and organs from harmful assaults in in vivo and in vitro experimental models. Hemorrhagic shock followed by resuscitation is the principal cause of death among trauma patients and soldiers in the battlefield. Although the underlying mechanisms are still not fully understood, it has been shown that nitric oxide (NO) overproduction and inducible nitric oxide synthase (iNOS) over-expression play important roles in producing injury caused by hemorrhagic shock including increases in polymorphonuclear neutrophils (PMN) infiltration to injured tissues and leukotriene B4 (LTB4) generation. Moreover, transcription factors responsible for iNOS expression are also altered by hemorrhage and resuscitation. It has been evident that either up-regulation of HSP-70i or down-regulation of iNOS can limit tissue injury caused by ischemia/reperfusion or hemorrhage/resuscitation. In our laboratory, geldanamycin, a member of ansamycin family, has been shown to induce HSP-70i overexpression and then subsequently to inhibit iNOS expression, to reduce cellular caspase-3 activity, and to preserve cellular ATP levels. HSP-70i is found to couple to iNOS and its transcription factor. Therefore, the complex formation between HSP-70i and iNOS may be a novel mechanism for protection from hemorrhage/resuscitation-induced injury.

Increased caspase-3 expression and activity contribute to reduced CD3ζ expression in systemic lupus erythematosus T cells

T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3ζ-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3ζ in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3ζ and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3ζ with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRγ-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3ζ-chain and suggest that such molecules can be considered in the treatment of this disease.

Selective inactivation of micro-organisms with near-infrared femtosecond laser pulses

We demonstrate an unconventional and revolutionary method for selective inactivation of micro-organisms by using near-infrared femtosecond laser pulses. We show that if the wavelength and pulse width of the excitation femtosecond laser are appropriately selected, there exists a window in power density that enables us to achieve selective inactivation of target viruses and bacteria without causing cytotoxicity in mammalian cells. This strategy targets the mechanical (vibrational) properties of micro-organisms, and thus its antimicrobial efficacy is likely unaffected by genetic mutation in the micro-organisms. Such a method may be effective against a wide variety of drug resistant micro-organisms and has broad implications in disinfection as well as in the development of novel treatments for viral and bacterial pathogens.

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+]i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 ± 2 nM (n = 23), indicating that [Ca2+] entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 ± 5% (P ≤ 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels. (Mol Cell Biochem 271: 51–59, 2005)