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.

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-alpha2 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.

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis.

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.

Thyroid injury, autoantigen availability, and the initiation of autoimmune thyroiditis.

Iodine depletion prevents disease and iodine repletion, which may cause thyroid cell injury by reactive oxygen intermediates, initiates disease in the Obese Strain chicken model of spontaneous autoimmune thyroiditis (AT). To examine the role of cell injury and autoantigen availability in AT induction we compared the immune responses that followed blunt trauma to the OS thyroid in the absence of iodine and the administration of normal dietary iodine in the absence of thyroid injury. Serum thyroglobulin concentrations were elevated following thyroid injury and the extensive thyroid infiltrates had high macrophage/CD4+, CD8+, B cell ratios consistent with an acute inflammatory response. The response was self limiting and undetectable in all animals 2 weeks later. Birds raised on a similar low iodine regimen were withdrawn from the regimen and given normal dietary iodine. Their thyroids showed no evidence of acute ultrastructural damage. The resulting early thyroid infiltrates had low macrophage/CD4+, CD8+, B cell ratios. Two weeks later these animals showed severe thyroid infiltration (43%) and after 4 weeks all animals had >90% infiltration. Thus, the presence or absence of thyroidal iodine, whether accompanied by injury or not, determined the nature and consequence of the immune response which argues against hypotheses that include obligatory injury at disease onset. Taken with previous work, this study suggests that iodination of an autoreactive thyroglobulin epitope is a requisite pathogenic action of iodine and supports the notion that in some organ-specific autoimmunity, a component of the dysregulation is associated with aberrant activity of the target tissue.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function.

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.

2'-deoxyadenosine induces apoptosis in rat chromaffin cells.

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 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and 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 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) 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.

Identification of tissue-specific DNase I hypersensitive sites in the rabbit flavin-containing monooxygenase form 2 gene.

Previous studies demonstrated that the flavin-containing monooxygenases (FMO) are expressed in a tissue-specific manner. To begin an elucidation of mechanisms regulating this expression pattern, genomic clones for rabbit FMO2 were isolated and characterized. Two clones were isolated from a lambda EMBL3 genomic library and shown to span approximately 30 kilobase pairs but to contain only about 800 base pairs of coding information. Primer extension analysis was used to map the transcription start site, extending the previously published cDNA sequence by 17 base pairs. The FMO2 promoter does not utilize a classical TATA box, nor are HTF islands present (DNA domains rich in cleavable sites for 5-methyl-cytidine/guanosine-sensitive restriction enzymes). Rather, homology with promoters controlled by initiator elements is observed. Previous studies demonstrated FMO2 expression in rabbit pulmonary Clara and type II cells [Overby, Nishio, Lawton, Plopper, and Philpot: Exp. Lung Res. 18, 131, (1992)]. In the present study, a highly enriched Clara/type II cell population was prepared, and the FMO2 gene was analyzed for DNase I-hypersensitive and methylated regions. These data were then contrasted with those obtained from a similar analysis of the nonexpressed, hepatocyte FMO2 gene. No difference in the methylation status was observed. However, Clara/type II cell-specific DNase I-hypersensitive sites are located within the promoter region of the FMO2 gene. Thus, tissue-specific transcription factors likely are more prominent than methylation in regulating FMO2 expression. Consistent with this observation, both polyomavirus enhancer activator 3 (E26 transformation specific) and thyroid transcription factor 1 consensus sequences are present within the tissue-specific DNase I-hypersensitive domain.

Epidermal growth factor-induced apoptosis in A431 cells can be reversed by reducing the tyrosine kinase activity.

A431 cells overexpress epidermal growth factor receptors (EGF-Rs) and are inhibited by EGF. We show that treatment of A431 cells with 10 nM EGF induced a 15-fold increase in EGF-R autophosphorylation, leading to inhibition of cell proliferation and morphological features of apoptosis. However, at a lower concentration of EGF (0.01 nM), there is a 2-fold increase in EGF-R autophosphorylation and increased cell proliferation when compared to untreated cells. EGF treatment is associated with increased expression of c-myc and decreased expression of mutant p53 and p21/WAF protein. When A431 cells were simultaneously treated with 10 nM EGF and EGF-R antibody, there was a significant reduction in EGF-R autophosphorylation that was associated with increased cell proliferation. Based on these results, we postulate that overexpression of EGF-R could allow for selective growth advantage for tumor cells in the presence of normal or decreased ligand availability. However, excessive ligand binding would result in deregulated growth signaling, leading to growth inhibition and programmed cell death.

Adenosine-induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine.

1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1-100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13-dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100-300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase-cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose-dependently inhibited by 5'-iodotubercidin (3-100 nM). 5'-Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2-deoxycytidine, but not by nicotinamide or 2-deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Deoxynucleoside induces neuronal apoptosis independent of neurotrophic factors.

Postmitotic sympathetic neurons are known to undergo a programmed cell death (apoptosis) when they are deprived of nerve growth factor (NGF) or treated with arabinofuranosyl nucleoside antimetabolites. Here we report the existence of a biochemical mechanism for the induction of neuronal death by an endogenous nucleoside in the presence of NGF. In support of such a mechanism we show that 2-deoxyadenosine (dAdo) induces apoptosis in chick embryonic sympathetic neurons supported in culture by NGF, excess K+, phorbol 12,13-dibutyrate, or forskolin. Neuronal death was related to a dramatic increase in the dATP content of sympathetic neurons exposed to dAdo (34.96 +/- 5.98 versus 0.75 +/- 0.16 pmol/micrograms protein in untreated controls, n = 9), implicating dATP in the toxicity. Supportive evidence for a central role of dATP was gained by inhibition of kinases necessary for phosphorylation of dAdo. 5'-Iodotubercidin in nanomolar concentrations completely and dose-dependently inhibited formation of dATP and also protected against toxicity of submillimolar concentrations of dAdo in sympathetic neurons. Although some of these actions of dAdo were remarkably similar to those reported for human lymphoid cells, several were uniquely different. For example, [3H]dAdo was not transported into neurons by the nucleoside transporter, and therefore inhibition of the transporter (dilazep, nitrobenzylthioinosine) did not prevent neurotoxicity by dAdo. Precursors of pyrimidine synthesis (2'-deoxycytidine, uridine) or NAD+ synthesis (nicotinamide) were ineffective in protecting sympathetic neurons against dAdo toxicity. Finally, inhibition of adenosine deaminase by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl) adenine did not potentiate the toxic effects of dAdo. Our results provide evidence for the first time that neuronal cells are as susceptible to nucleoside lethality as human lymphocytes are, and provide a new model to study the salvage pathway of deoxyribonucleosides in controlling neuronal populations through programmed cell death.

Vitamin D3 receptor expression in N-ethylnitrosourea-induced mouse pulmonary adenomas.

An expanded role for vitamin D (1 alpha,25-(OH)2D3) in mammalian systems has been suggested by recent evidence that its receptor (vitamin D receptor [VDR]) is present not only in classical target organs, but in a variety of normal tissues and organs, tumor tissues, and cancer cell lines. Vitamin D is involved not only in the regulation of calcium homeostasis and bone metabolism, but in the regulation of cell proliferation, differentiation, and immune responses. The role vitamin D may play in normal lung growth, development, and maintenance is unknown. Likewise, its part in lung tumorigenesis is unclear. The present study examined VDR binding activity and VDR expression in normal mouse lung and ethylnitrosourea-induced lung adenomas. Binding of 1 alpha,25-(OH)2D3 was specific and saturable over the concentration range of 0.01 to 0.50 nM, with an affinity (Kd) of 0.93 x 10(-10) M and a total binding capacity (Bmax) of 22 fmol/mg of protein. Scatchard analysis yielded a convex curve, which suggests positive receptor cooperativity. The calculated Hill coefficient equals 1.69, at a receptor concentration of 0.4 nM, consistent with dimerization of the receptor. Western blot analysis showed the presence of 60 kD VDR protein in tumor homogenates, while Northern blot analysis detected the 4.4 kb VDR mRNA in tumor tissue preparations. Immunohistochemistry and in situ hybridization revealed that both adenomatous Clara cells and normal bronchiolar epithelial Clara cells expressed VDR, with the receptor protein present in their nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Amyloid plaque cores were purified from Alzheimer disease brain tissue. Plaque core proteins were solubilized in formic acid which upon dialysis against guanidinium hydrochloride (GuHCl) partitioned into soluble (approximately 15%) and insoluble (approximately 85%) components. The GuHCl-soluble fraction contained beta-amyloid1-40, whereas the GuHCl-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 GuHCl 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 beta-amyloid. Immunochemical analyses revealed alpha 1-antichymotrypsin and non-beta-amyloid segments of the beta-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 fluorescein isothiocyanate- and colloidal gold-conjugated lectins. We have shown previously that this plaque core protein complex is more toxic to neuronal cultures than beta-amyloid. The non-beta-amyloid components likely mediate this additional toxicity, imposing a significant influence on the pathophysiology of Alzheimer disease.

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.

Shigella dysenteriae 60R strain adheres to and invades tissue culture cells in the absence of virulence plasmid.

Evidence is presented that a high level Shiga toxin-producing strain Shigella dysenteriae 60R adheres to and invades the epithelial cell lines Hct8 and Henle 407. The invasive phenotype of S. dysenteriae 60R differs in four ways from the heretofore studied invasive Shigella phenotypes. First, S. dysenteriae 60R lacks the virulence plasmid characteristic of other invasive Shigella spp. and enteroinvasive Escherichia coli. Second, hybridization studies show that the known ipa genes are neither present in the chromosome nor in the small plasmid of 60R. Third, 60R adheres to and invades Hct8 and Henle 407 cells at 37 degrees C as well as at 30 degrees C. Fourth, the phenotype of adherence and invasion of 60R is remarkably stable, even during prolonged growth in laboratory media and storage.

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

Purification of amyloid plaque core proteins (APCP) from Alzheimer's 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.

Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action.

Thymosin alpha 1 (T alpha 1), the N-terminal 28-amino acid fragment of prothymosin alpha (ProT alpha), and ProT alpha, although originally isolated from whole thymus extracts, are also present in nonthymic cells and tissues. We used an ELISA with an antibody raised against T alpha 1 to investigate the relationship between intracellular levels of thymosin immunoreactive peptide(s) (TIP) and cell proliferation in a rat small intestinal IEC-6 cell line. Increasing TIP levels were observed during cell proliferation, which decreased when proliferation was halted by cellular contact inhibition. Serum feeding of cells previously rendered quiescent by serum starvation resulted in a significant increase in TIP within 1 hr. Conversely, serum starvation decreased TIP levels within 1 hr. Peak TIP levels appeared after 3 hr of serum incubation, while maximum [3H]thymidine incorporation was noted after 9 hr, suggesting maximum TIP concentrations in the G1 phase of the proliferative cycle. Immunoelectron microscopy demonstrated an association of TIP with condensed nuclear chromatin. These results support a relation of intracellular TIP levels to IEC-6 cell proliferation and also a nuclear site of action. HPLC analysis of cellular homogenates from proliferating IEC-6 cells revealed a peak of immune reactivity that elutes in the position of T alpha 1.

Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.

The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.

Inhibition of rat ovarian [3H]thymidine uptake by luteinizing hormone-releasing hormone agonists: a possible mechanism for preventing damage by cytotoxic agents.

To investigate the mechanism involved in luteinizing hormone-releasing hormone (LHRH) agonists' protective effects against chemotherapy-induced ovarian damage, female rats were either implanted with 1-mg pellets of LHRH agonist Zoladex (LHRHz) or sham operated. All rats were implanted with osmotic minipumps loaded with [3H]thymidine 48 h before sacrifice in diestrus. Ovaries were combusted in a biological material oxidizer. Tritiated water was recovered in a special cocktail, and ovarian tritiated thymidine uptake (3HTU) was calculated. In five experiments, LHRHz significantly reduced ovarian 3HTU. This was observed 5 days after implanting LHRHz pellets. Ovarian 3HTU correlated significantly with serum estradiol, LH, and ovarian and uterine weights. Autoradiography showed that almost all ovarian 3HTU is by granulosa cells. These data suggest that LHRHz suppresses ovarian mitotic activity. Since cytotoxic agents preferentially destroy rapidly dividing cells, our findings may represent a mechanism for ovarian protection.

An animal model of pulmonary radiation fibrosis with biochemical, physiologic, immunologic, and morphologic observations.

An animal model of pulmonary radiation fibrosis was established, using male CBA/j mice. Both lungs of each mouse in one group (DL) were irradiated with two doses of 8.5 Gy each, separated by 30 days. A control group (CG) was sham-irradiated. There was a small but significant difference (P less than 0.03) in average breathing rate between DL and CG 27 weeks after the second irradiation which increased until the 34th week followed by a plateau. The accumulated hydroxyproline content of the irradiated mouse lung was 40% greater (P less than 0.02) than that of the sham-irradiated lung at 42 weeks and thereafter. Anticollagen antibodies assayed 52 weeks after irradiation by enzyme-linked immunosorbent assay were elevated by 49% in sera from the irradiated mice compared to sera from sham-irradiated mice. Mortality during the 52-week period following the second irradiation was low (13%) for both groups. Histological comparison of irradiated and control mouse lungs fixed under uniform inflation pressure indicated no significant differences. The model has unique features including an increase in collagen deposition, no acute changes attributable to radiation, a small but statistically significant abnormality in pulmonary function, an immunologic response to collagen, and low mortality.