Kenneth C. Palmer

Morphological and biochemical analyses of amyloid plaque core proteins purified from Alzheimer disease brain tissue

Abstract— Amyloid plaque cores were purified from Alzheimer disease brain tissue. Plaque core proteins were solubilized in formic acid which upon dialysis against guan‐idinium hydrochloride (GuHCI) partitioned into soluble (∼15%) and insoluble (∼85%) components. The GuHCI‐soluble fraction contained β‐amyloid1‐40, whereas the GuHCI‐insoluble fraction was fractionated into six components by size exclusion HPLC: S1 (>200 kDa), S2 (200 kDa), S3 (45 kDa), S4 (15 kDa), S5 (10 kDa), and S6 (5 kDa). Removal of the GuHCI reconstituted 10‐nm filaments composed of two intertwined 5‐nm strands. Fractions S5 and S6 also yielded filamentous structures when treated similarly, whereas fractions S1–S4 yielded amorphous aggregates. Chemical analysis identified S4–S6 as multimeric and monomeric β‐amyloid. Immunochemical analyses revealed α1‐antichymotrypsin and non‐β‐amyloid segments of the β‐amyloid precursor protein within fractions S1 and S2. Several saccharide components were identified within plaque core protein preparations by fluorescence and electron microscopy, as seen with fluores‐cein isothiocyanate‐and colloidal gold‐conjugated lectins. We have shown previously that this plaque core protein complex is more toxic to neuronal cultures than β‐amyloid. The non‐β‐amyloid components likely mediate this additional toxicity, imposing a significant influence on the pathophysiology of Alzheimer disease.

Differential regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression in co-cultures of prostate cancer and stromal cells

Tumor–stromal interactions have been suggested to be a critical factor in both tumor invasion and tumor metastasis. Here, we examined the role of tumor–stromal interactions using co‐cultures of prostate cancer (PC) cells derived from primary and metastatic tumors with primary or immortalized stromal (fibroblast and smooth muscle) cells and their effect on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression. Co‐cultures of PC and stromal cells showed enhanced levels of pro‐MMP‐9 and reduced levels of TIMP‐1 and TIMP‐2. Whereas enhanced expression of pro‐MMP‐9 occurred in PC cells, the TIMPs were down‐regulated in stromal cells. Induction of pro‐MMP‐9 and reduction of TIMP expression did not require cell–cell contact and were mediated by a soluble factor(s) present in the conditioned medium of the effector cell. Collagen I is a potent inducer of pro‐MMP‐9 in PC cells. Consistently, preliminary characterization of the soluble factor in the fibroblast conditioned medium revealed m.w. of approximately 100 to 250 kDa, and its effect on pro‐MMP‐9 expression was partly inhibited by an anti‐α2 integrin antibody, a major collagen I receptor. Expression of pro‐MMP‐9 protein and mRNA was also induced in metastatic PC‐3 cells grown in human fetal bone implants in SCID mice. Together, these findings demonstrate the importance of tumor–stromal interactions in the regulation of MMP and TIMP expression and their potential role in PC progression.

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.

Calcium blockade reduces renal apoptosis during ischemia reperfusion

Apoptosis is well described in invertebrates and recently documented in mammals. The prevalence and pathophysiology of mammalian apoptosis is unknown and may have clinical ramifications. The aim of this study is to investigate the apoptotic response during kidney ischemia-reperfusion (I/R) injury. Kidney I/R was initiated in anesthetized rats by occlusion of the renal pedicle for 45 min with or without pretreatment with .2 mg/kg verapamil: control animals received sham exposure. Flow was re-established after ischemia and the animals were allowed to recover for 24 h. Bilateral kidneys were harvested for DNA electrophoresis, Western analysis for p53, Northern analysis for c-myc expression, and light and electron microscopic analysis. Kidney I/R caused characteristic DNA laddering in the clamped kidney, and less extensive laddering was seen in the contralateral kidney. Light and electron microscopic analysis confirmed apoptotic morphology in the reperfused tissues. Verapamil pretreatment completely abolished DNA laddering and attenuated the microscopic evidence of apoptosis. p53 levels were increased by I/R in the ischemic kidney and moderately increased in the contralateral organ, c-myc mRNA levels were increased by the I/R insult. Kidney I/R injury may induce global apoptosis, which seems to be associated with an alteration in calcium homeostasis. The increase in p53 and c-myc mRNA levels seen with I/R may facilitate apoptosis. Calcium modulation seems to reduce apoptosis during I/R and may have therapeutic implications.

In-vivo particle mediated delivery of mRNA to mammalian tissues: Ballistic and biologic effects

Biolistic transmission of mRNA provides transient gene therapy to in vivo organs. This study documents particle mediated mRNA transmission to a solid organ and wound healing model using the mRNA of Green Fluorescent Protein to determine optimal delivery parameters. Renal function, bullet penetration, cellular injury, and Green Fluorescent Protein synthesis were quantified. Chimeric human epidermal growth factor‐FLAG epitope cDNA or mRNA was transmitted to wounds in normal or steroid treated animals. Wound bursting strength, human epidermal growth factor‐FLAG, and collagen synthesis were determined. Injury and bullet penetration correlated with the delivery velocity and bullet size. Optimal delivery parameters were established which provided widespread Green Fluorescent Protein synthesis. Human epidermal growth factor‐FLAG treatment significantly increased collagen content and wound breaking strength in normal and steroid treated animals. FLAG protein synthesis was evident in mRNA treated fascia following treatment. We found the gene gun provides a novel method for efficient, in vivo delivery of mRNA‐based therapeutic strategies to mammalian organs with minimal histologic damage allowing transient expression of protein in in vivo target tissues. Co‐delivery of Green Fluorescent Protein mRNA may provide a useful positive control to determine effective transmission. Biolistic transmission of human epidermal growth factor‐FLAG mRNA provides increased tissue epidermal growth factor levels and accelerates wound healing in normal and steroid exposed animals.

New biochemical insights to unravel the pathogenesis of Alzheimer's lesions.

Purification of amyloid plaque core proteins (APCP) from Alzheimers disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of beta-amyloid (beta A) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N- and O-linked saccharides. When added to tissue culture of sympathetic and sensory neurons APCP and beta A inhibited neuritic sprouting, a reversible phenomenon at low doses. Higher concentrations of both substances kill the neurons in culture. APCP is significantly more toxic than beta A, suggesting the minor components may play an important role in increasing the toxicity of beta A. If the observed toxic effects of APCP in situ are occurring in vivo during the course of AD, then the accumulation of these extracellular proteins could be largely responsible for some of the neuronal death observed in this neuropathology.

2'-deoxyadenosine induces apoptosis in rat chromaffin cells.

Abstract: We show here that 2′‐deoxyadenosine (2′‐dAdo) but not adenosine was toxic to chromaffin cells of 3–4‐week‐old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3‐day period in the presence of 100 µM 2′‐dAdo plus 3 µM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase‐mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis‐like cell death by 2′‐dAdo. Lethal effects of 2′‐dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2′‐dAdo‐prompted cell death was not prevented by inhibitors of nucleoside transporter (3 µM dilazep or 1 µM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 µM uridine or 100 µM 2′‐deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2′‐dAdo (100 and 300 µM) showed a three‐ and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2′‐dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2′‐dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.

Cadmium-induced acute lung injury: Compromised repair response following thyroidectomy

The role of thyroid hormone in the pulmonary repair process following chloride-induced acute injury, was assessed in the present study. Thyroidectomized (Thyx), male Sprague-Dawley rats were exposed by inhalation to cadmium chloride aerosol (CdCl2, 10 mg/m3). Rats were sacrificed 1 hr after [3H]thymidine (3H-T) injection at intervals up to 10 days after exposure. Thyroidectomy, followed by CdCl2, produced earlier and more severe acute injury in the form of alveolar hemorrhage edema and hyaline membrane formation, than CdCl2 alone. However, Type 2 cell hyperplasia was markedly reduced in this group of rats compared with CdCl2 controls. Uptake of 3H-T by Thyx-CdCl2 lung tissue was only 40% of that measured in CdCl2 controls. Autoradiographic studies indicated that Type 2 cell labeling was less than 66% of controls up to 3 days after exposure. Cells obtained by lung lavage of Thyx-CdCl2 rats were reduced in number up to 60% with respect to controls, during the first week after exposure. Additionally, the activities of lung antioxidant enzymes (glucose-6-phosphate dehydrogenase, superoxide dismutase, and glutathione peroxidase were significantly inhibited (45-55%) throughout the experiment in Thyx-CdCl2 animals compared with normal rats. In summary, thyroidectomy impairs the repair response in CdCl2 lung damage by enhancing Type 2 cell damage, reducing Type 2 cell proliferation, altering alveolar macrophage populations, and depressing antioxidant defense systems.

Lung aryl hydrocarbon hydroxylase: Inhibition following cadmium chloride inhalation

Polycyclic hydrocarbon metabolism in male Sprague-Dawley rats following inhalation of aerosolized cadmium chloride (CdCl2) was examined. Constitutive activity of microsomal aryl hydrocarbon hydroxylase (AHH) (benzo(a)pyrene substrate) was monitored in lung and liver homogenates up to 10 days after exposure. Lung AHH activity was reduced by 85% during the first 2 days following cadmium inhalation, and did not return to normal levels until 7 days after exposure. Enzyme activity in the livers of cadmium-treated animals was similarly depressed (by 65%) within 24 hr. Cadmium inhalation also inhibited (by 50%) 3-methylcholanthrene (MC) induction of lung AHH when compared with MC-treated controls. No significant effect on AHH inducibility by MC was noted in liver homogenates from cadmium-exposed animals. Nonspecific microsomal damage appeared not to occur since glucose-6-phosphatase activity in lung was unaffected by cadmium treatment. Although the mechanism of cadmiums action remains unclear, it appears not to involve a direct interaction of the metal with enzyme. The alteration of AHH activity by cadmium may result from injury to a specific cell type within the lung, which may be a major site of pulmonary AHH activity, or may result from modulation of synthesis and/or degradation of heme proteins in the lung. These results suggest that cadmium, under these conditions, markedly reduces the constitutive and inducible activity of AHH in the lung.