The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

A Fluorescent Anti-Cancer Agent, 3,6-bis(1-methyl-4-vinylpyridinium) Carbazole Diiodide, Stains G-Quadruplexes in Cells and Inhibits Tumor Growth.

Compelling evidence suggests that formation of guanine-quadruplex (G4) can protect the integrity of chromosome ends in eukaryotes, and regulate the activity of some gene promoters. In addition, G4 may be a novel therapeutic target. Thus, a number of ligands have been synthesized to stabilize G4. However, skepticism lingers over the existence of G4 in cells, as well as its biological function. The molecule 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) can be used not only as a fluorescent probe to map endogenous and exogenous G4 in live cells, but also as therapeutic agent that arrests cancer growth by inhibiting telomerase activity and regulating gene expression. Thus, the fluorescence of a G4 anti-cancer agent is an invaluable tool to detect G4 in cells, investigate ligand-G4 interaction in live cells, examine the biological function of G4, and guide the development of new fluorescent anti-cancer agents.

Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy.

The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8 ns and that of interaction with the duplex structure of a calf thymus is ∼1.2 ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

Massive calcium-activated endocytosis without involvement of classical endocytic proteins.

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief ß-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells.

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5-10 microg ml(-1)), BFA caused both the Golgi apparatus and trans-Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY-2 cells. Here we show that these BFA-induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA-induced Golgi- and TGN-derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4-64 co-localized with the TGN-derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose- and time-dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4-64 are mostly excluded from the SYP61-positive BFA-induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.

Bone quality determined by Fourier transform infrared imaging analysis in mild primary hyperparathyroidism.

CONTEXT: Mild primary hyperparathyroidism (PHPT) is characterized by asymptomatic hypercalcemia, most commonly in the absence of classical signs and symptoms. Hence, there is need to characterize this disorder with particular attention to the skeleton. DESIGN: We determined the ratio of pyridinium and dehydrodihydroxylysinonorleucine collagen cross-links in 46 iliac crest bone biopsies from patients with PHPT (14 men, aged 28-68 yr; 32 women, aged 26-74 yr) by Fourier transform infrared imaging. The results were compared with previously reported collagen cross-links ratio determined in iliac crest biopsies from normal subjects. RESULTS: PHPT patients exhibited significantly lower pyridinium to dehydrodihydroxylysinonorleucine collagen cross-links ratio, compared with normal controls. Parathyroidectomy restored values to those comparable with normal controls. Moreover, the differences among PHPT subjects were gender dependent, with female PHPT patients having a statistically significant lower ratio, compared with either male PHPT patients or normal controls. Comparison of the obtained outcomes with histomorphometry showed that the collagen cross-link ratio was strongly correlated with rate of bone formation, and mineralizing surface, in individual patients. This ratio was also correlated with bone mineralization density distribution parameters obtained in the same patients. The strongest correlations were with bone mineralization density distribution variables reflecting heterogeneity of mineralization and primary mineralization parameters. CONCLUSIONS: The results are consistent with the high turnover state manifested in PHPT patients. Reduced collagen cross-link ratio in patients with PHPT would be expected to reduce the stiffness of bone tissue. These observations provide a more complete assessment of bone material properties in this disorder.

IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri.

The effects of several hormones on pollen tube growth were compared in Torenia fournieri and it was found that IAA was the most effective, stimulating pollen tube growth and causing the shank part of pollen tubes to be slender and straighter. The role of IAA was investigated by studying the changes in ultrastructure and PM H(+)-ATPase distribution in the pollen tubes and the modification of the tube wall. Using the fluorescent marker FM4-64, together with transmission electron microscopy, it was shown that secretory vesicles and mitochondria increased in IAA-treated tubes. Immunolocalization and fluorescence labelling, together with Fourier-transform infrared analysis, detected that IAA enhanced the level of PM H(+)-ATPase and the synthesis of pectins, and reduced the cellulose density in pollen tubes. Importantly, to observe the orientation of cellulose microfibrils in pollen tubes in situ, atomic force microscopy was used to examine the 'intact' tube wall. Atomic force microscopy images showed that cellulose microfibrils were parallel to each other in the subapical region of IAA-treated tubes, but disorganized in control tubes. All results provided new insights into the functions of cellulose microfibrils in pollen tube growth and direction, and revealed that the IAA-induced changes of pollen tubes were attributed to the increase in secretory vesicles, mitochondria, and PM H(+)-ATPase, and the modification of pectin and cellulose microfibrils in the tube wall.

Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells.

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.

Application of hydrophilic interaction liquid chromatography/comparative taste dilution analysis for identification of a bitter inhibitor by a combinatorial approach based on Maillard reaction chemistry.

Activity-directed fractionation of heated carbohydrate/alanine solutions recently led to the discovery of (+)-(S)-1-(1-carboxyethyl)-5-hydroxy-2-(hydroxymethyl)pyridinium inner salt (1, alapyridaine), and it has been shown that this compound lowers the detection thresholds of sugars, glutamate, and NaCl solutions, whereas no influence on bitter perception was observed. As this class of Maillard-derived pyridinium betaines seemed to be promising targets for further research on their taste modulatory activity, the objective of the present investigation was to screen for bitter taste-suppressing target molecules in combinatorial libraries of pyridinium betaines prepared from 5-(hydroxymethyl)furan-2-aldehyde and amino acid mixtures by use of Maillard-type reaction chemistry instead of synthesizing and purifying each derivative individually. By application of hydrophilic interaction liquid chromatography in combination with the recently developed comparative taste dilution analysis, followed by structure determination, synthesis, and sensory studies, we have now succeeded in identifying 1-carboxymethyl-5-hydroxy-2-hydroxymethylpyridinium inner salt (2) as a potential bitter-suppressing candidate. While tasteless on its own, 2 was found to reduce the bitterness of various bitter tastants such as the amino acid L-phenylalanine, the peptide Gly-Leu, the alkaloid caffeine, and the bitter glycosides salicin and naringin.

Activity-guided identification of a chemopreventive compound in coffee beverage using in vitro and in vivo techniques.

The aim of the present study was to apply an activity-guided screening procedure to coffee brew to identify a key chemopreventive compound by means of in vitro antioxidant tests as well as cell culture experiments and to prove the in vivo activity of that compound by an animal feeding experiment. Solvent fractionation, followed by multiple-step ultrafiltration, revealed that the polar coffee compounds with molecular weights below 1 kDa show the major inhibitory effect on the in vitro peroxidation of linoleic acid as well as the predominant chemopreventive enzyme modulating activity on the NADPH-cytochrome c reductase (CCR) and glutathione S-transferase (GST) in human intestinal Caco-2 cells. To identify the chemical structure of the most active antioxidants and chemopreventive compounds, the polar compounds were further separated by HPLC techniques, followed by the activity-guided screening of the individual HPLC fraction. These experiments demonstrated 5-chlorogenic acid to be the most powerful antioxidant in vitro, whereas, in contrast, chemopreventive effects on the GST activity were found for the N-methylpyridinium ion, the structure of which was elucidated by LC-MS and NMR experiments and confirmed by synthesis. The in vivo activities of coffee beverage and N-methylpyridinium ions were tested in a 15-day feeding experiment on rats. In the liver, feeding of 4.5% coffee beverage resulted in increases of GST and UDP-GT activities by 24 and 40% compared to animals fed the control diet (p > 0.05), respectively. Plasma total antioxidant capacity and plasma tocopherol were elevated in animals fed the coffee beverage and the N-methylpyridinium-containing diet. In summary, the results demonstrating a strong in vitro antioxidant activity for coffee were confirmed by the feeding study. Surprisingly, feeding of N-methylpyridinium also resulted in an increased total antioxidant capacity in the plasma. The data indicate that the mode of action demonstrated for N-methylpyridinium in biological systems is different from that in foods.

Dorsal and palmar material properties of the adult human flexor profundus tendon in Zone II.

Nineteen fresh frozen adult human flexor digitorum profundus (FDP) tendons in Zone II were studied to compare the differences in material properties between the dorsal (dFDP) and palmar (pFDP) side of each tendon biomechanically, biochemically and histologically. We have found that tissue from the dorsal side of each flexor tendon has (1) greater strength; (2) less collagen crosslinking (hydroxypyridinium); and (3) a larger single bundle cross-sectional area than tissue from the palmar side of the same tendon. These data clearly demonstrate that the dorsal and palmar sides of the adult human (FDP) tendon in Zone II differ materially. These differences suggest that there may be biomechanical advantages in placing core sutures dorsally when repairing flexor tendons, a technique that we have previously described.

Alteration of elastin, collagen and their cross-links in abdominal aortic aneurysms.

OBJECTIVES: although the mechanism of arterial dilation and aneurysm development has not been clarified, the degradation of elastin and collagen plays undoubtedly a critical role. We evaluated the elastin and collagen content through the detection of their cross-links in aneurysmal and non-aneurysmal abdominal aortic walls. MATERIALS AND METHODS: in 26 human abdominal aortic aneurysm specimens obtained during surgery and in 24 autopsy control samples of non-aneurysmal abdominal aorta the tissue content of elastin and collagen cross-links were measured by HPLC. Collagen was also detected by evaluating two characteristic amino acids, 4-hydroxyproline (4-hypro) with a colorimetric method and 5-hydroxylysine (5-hylys) by gas chromatography. RESULTS: significantly fewer elastin cross-links were found in aneurysm samples compared to controls (desmosines and isodesmosines: 90% reduction; p<0.01). The opposite was true for pyridinoline collagen cross-links (350% increase) and deoxypyridinolines (100% increase, p=0.01). Tissue content of 5-hylys, 4-hypro and total amino acids were reduced significantly by 50% in aneurysmal samples. CONCLUSIONS: beside confirming decreased elastin content in aneurysmal walls, these results show a concurrent increase of collagen cross-links. Since total collagen markers were decreased (decreased 4-hypro and 5-hylys) it is reasonable to suggest that in aneurysmal aortic walls old collagen accumulates cross-links while new collagen biosynthesis is somehow defective.

Effect of estrogen deficiency on skeletal and alveolar bone density in sheep.

BACKGROUND: This study provides a longitudinal assessment of changes in alveolar and skeletal bone mineral density (BMD) in ovariectomized animals. METHODS: Following ovariectomy (OVX) (n = 6) or sham-operation (n = 6) intraoral radiographs were made at 4-month intervals and serum 17-beta-estradiol, osteocalcin, and interleukin (IL)-6, urinary deoxypyridinium, and salivary IL-6, deoxypyridinium, and osteocalcin concentrations were evaluated. Twelve months after surgery, animals were sacrificed and the mandible and radius/ulna removed. Bones were sectioned and radiographed. Mean BMD and cortical thicknesses were calculated from each region. RESULTS: OVX animals had a progressive decrease in serum 17-beta-estradiol, increased serum osteocalcin and IL-6, urinary deoxypyridinium and salivary IL-6, osteocalcin and deoxypyridinium (P < 0.001), suggesting that they were becoming osteoporotic. The BMD of the radius/ulna and mandibular alveolar bone was significantly reduced in OVX animals (P < 0.05 and P < 0.001, respectively). Reduced alveolar bone BMD became evident in OVX animals 6 months after surgery and became more severe during the subsequent 6 months. Alveolar crestal height was also significantly reduced in OVX animals (P < 0.001). These biochemical and density changes preceded a significant reduction in serum 17-beta-estradiol, which occurred between 4 and 8 months following surgery. CONCLUSIONS: Serial measurements of alveolar BMD predicts loss of skeletal BMD in OVX sheep. Changes in alveolar BMD precede estrogen deficiency, suggesting that early signs of reduced BMD may be detected in peri-menopausal women. The presence of biomarkers of bone metabolism within saliva and their correlation with reduced BMD suggests that saliva could be used as an adjunct screening method for assessment of skeletal bone density.

High-performance liquid chromatographic assays for a second-generation novel oral iron chelator (APCP363) and their application to pharmacokinetic studies in rats.

Sensitive and specific HPLC assays for APCP363 in biological matrices (rat plasma, urine and feces) were developed. The recovery of APCP363 ranged from 81.2 to 99.9% in plasma, from 82.1 to 92.8% in urine, and from 65 to 68% in feces. Standard deviations were below 10% for all analyses. The limits of quantitation were 0.1, 10 and 30 microg/ml in plasma, urine and feces, respectively. The HPLC assays, which are the first reports for APCP363 analysis in biological matrices, have been successfully applied to preliminary pharmacokinetic studies in rats. The stool assay is the first non-radiolabeled method for hydroxypyridinones in feces.

Electrogenic properties of the Na+,K+-ATPase probed by presteady state and relaxation studies.

Electrical measurements on planar lipid bilayers, patch/voltage clamp experiments, and spectroscopic investigations involving a potential sensitive dye are reviewed. These experiments were performed to analyze the kinetics of charge translocation of the Na+,K+-ATPase. High time resolution was achieved by applying caged ATP, voltage-jump, and stopped-flow techniques, respectively. Kinetic parameters and the electrogenicity of the relevant transitions in the Na+,K+-ATPase reaction cycle are discussed.

Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.

Comparison of the clinical performance of the immunoenzymometric assays for N-terminal and C-terminal type I collagen telopeptides and the HPLC assay for pyridinium cross-links.

We evaluated the clinical performances of the immunoenzymometric assays for type I collagen N-terminal and C-terminal telopeptides and the HPLC assay for total deoxypyridinoline, in distinguishing between subjects with a moderately increased bone resorption rate (women in postmenopause) and subjects with normal bone resorption rate (women in premenopause). The postmenopausal group consisted of 61 women who had been in menopause for no more than 10 years, while the premenopausal group consisted of 52 healthy women with normal menstrual cycles. The biochemical markers were measured in a 24 hour urine sample and the results expressed as the molar ratio with urinary creatinine. The clinical performances were estimated by calculating the accuracy (as the area under a Receiver Operated Characteristic (ROC) curve: mean +/- SEM) and the discriminating power (as score) of each assay in distinguishing postmenopausal subjects from premenopausal subjects. Type I collagen C-terminal telopeptide, type I collagen N-terminal telopeptide and total deoxypyridinoline were significantly higher in the postmenopausal than in the premenopausal group (p < 0.01). Accuracies of these three markers ranged from 66.8 +/- 5.1% to 76.8 +/- 4.3%, while Z scores ranged from 3.54 to 5.67. Type I collagen C-terminal telopeptide, type I collagen N-terminal telopeptide and total deoxypyridinoline were not significantly different in their accuracy or discriminating power. All markers were highly correlated with coefficients of correlation ranging from 0.61 to 0.77. In summary, this study shows that 1) the immunoenzymometric assays for type I collagen N-terminal telopeptide and type I collagen C-terminal telopeptide show a high accuracy and discriminating power in distinguishing subjects with different bone resorption rate; 2) the results obtained with these immunoenzymometric assays are comparable to those obtained with the HPLC assay for total deoxypyridinoline. In conclusion our data support the use of the immunoenzymometric assays for type I collagen N-terminal telopeptide and type I collagen C-terminal telopeptide for estimating bone resorption.

Lysyl oxidase expression and collagen cross-linking during chronic adriamycin nephropathy.

Collagen cross-linking induced by lysyl oxidase has been implicated in liver and lung fibrosis. To define the role of this process in kidney fibrosis, we investigated the renal expression of lysyl oxidase and the content in collagen cross-links at various stages of chronic Adriamycin nephropathy in Sprague-Dawley rats. Lysyl oxidase expression was determined by RT-PCR; collagen pyridinium residues, indicating lysyl oxidase induced cross-links, were evaluated by HPLC. These parameters followed a synergic albeit asynchronous outcome: (a) lysyl oxidase mRNA levels in total kidney, glomeruli and medulla from Adriamycin-treated rats increased up to 3 times compared to controls between week 8 and 12, then returning within the normal range; (b) the pyridinium residue content did not show any significant difference between Adriamycin-treated and control rats, until diffuse interstitial fibrosis developed (16 weeks), showing at this time a 2- to 3-fold increment. Lysyl oxidase was expressed by several renal cell lines and in tubular-epithelial cells it was up-regulated in vitro by TGF beta-1, a recognized fibrogenetic factor in Adriamycin nephropathy. Our observations demonstrated that an increased expression of lysyl oxidase in the kidney precedes the development of diffuse fibrotic lesions and that, at this stage, collagenic structures contain highly cross-linked components, the final product of lysyl oxidase activity. The evidence of lysyl oxidase up-regulation in tubular epithelial cells by the same factor implicated in Adriamycin toxicity in the kidney suggests a common pathogenetic mechanism. Collagen cross-link formation by lysyl oxidase may be implicated in the pathogenesis of irreversible, fibrotic renal lesions.

Acute pneumocyte injury, poly(ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide.

Cyclophosphamide (CYC) is a metabolically activated, DNA-alkylating, antitumor agent that causes pulmonary fibrosis. BALB/cN (B) mice are sensitive and C57Bl/6N (C) mice are resistant to CYC-induced fibrosis. Pulmonary bioactivation may contribute to strain sensitivity. Therefore, we tested the intrinsic susceptibility of murine lung slices to cell injury by direct exposure to CYC for 2-8 hr. Injury was measured by release of lactate dehydrogenase (LDH). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP, EC 2.4.2.30), causing depletion of its substrate, NAD. NAD can also be decreased by phosphorylation to NADP, as seen with oxidative stress. Depletion of NAD can lead to loss of ATP. Thus, we measured LDH release, PAP activation, NAD, NADP and ATP in slices incubated with or without the PAP-inhibitor, 3-aminobenzamide (3-AB). CYC (0.1 to 1.0 mg/mL for 4-8 hr) caused LDH release in slices from both murine strains, but LDH release was significantly greater in B lung slices than in C slices. After an 8-hr incubation 63.9 +/- 3.7% (mean +/- SEM) of total LDH was released from B lung slices with 1.0 mg CYC/mL, whereas only 45.8 +/- 2.6% was released from C lung slices (P < 0.05). 3-AB reduced LDH release to 44.7 +/- 2.4% in B slices and 28.1 +/- 2.0% in C slices (P < 0.05 vs CYC only). PAP activity in nuclei isolated from CYC-treated B lung slices was increased 2- to 4-fold after 2 hr of incubation with 0.5 and 1.0 mg CYC/mL. PAP activation was delayed and reduced with incubation in 3-AB. PAP was activated 2-fold in nuclei from C slices treated with 0.5 mg CYC/mL for 2 hr. NAD was decreased at 2 and 4 hr in B slices treated with 0.5 and 1.0 mg CYC/mL, and at 4 hr with 0.1 mg CYC/mL. NAD depletion occurred only at 4 hr in the resistant C slices treated with 1.0 mg CYC/mL. CYC increased NADP by a similar extent in B and C lung slices. In B slices, NAD losses were approximately 4 times the increases in NADP. CYC did not decrease ATP in B slices and ATP dropped 25% only after 4 hr in the resistant C slices. We conclude that CYC is directly toxic to lung tissue and observe that strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo. PAP activation and oxidative stress may contribute to this toxicity.