[Morphological-functional characteristic of periarticular tissue after total hip arthroplasty: histological, cytochemical and electron microscopy aspects]. / Morfologicko-funkcná charakteristika periartikulárneho tkaniva po endoprotéze bedrového klbu: histologické, cytochemické a elektrónmikroskopické aspekty.

PURPOSE OF THE STUDY Analysis of the tissue harvested around the total hip replacement isolated from re-operated patients in order to: (1) characterize complexity of structural processes developing in the region of the total hip replacement and, (2) to define the role and significance of histological structures of this tissue, mainly in relation to implant loosening, from the viewpoint of formal and causal pathogenesis. MATERIAL AND METHODS Biopsied material isolated from periarticular tissue of re-operated patients (n=19) after THR was analyzed using the methods of light optic, fluorescent (TUNEL), and transmission electron microscopy. RESULTS Histological analysis revealed fibroproliferaive processes and epithelioid granulomatosis cell reaction around the implant with the formation of giant multinuclear syncythial (osteoclastlike) structures as a response to foreign bodies. These structures phagocyte fragments of foreign material (polyethylene particles from the implant, cement fragments). All the used methods revealed a range of regressive changes in the layers of foreign microparticles (inside giant multinuclear cells) typical of fibrinoid necrosis in collagen fibres and apoptosis. In certain cases, progressive changes as chondroid and synovial differentiation (metaplasia) were observed. DISCUSSION Total hip replacement, despite all positive aspects for patients, may cause permanent inflammatory processes in its surrounding. This may result in an extensive fibroproduction of a differently thick layer of connective tissue around the implant. An important factor of loosening of THR is probably osteoclastic resorption in the area of "bone-implant" interface, as a result of the interaction between the inflammatory mechanisms around the implant. CONCLUSION In the postoperative period, there occur fibroproliferative changes in the periarticular tissue and large population of multinuclear cells. In our view, these cells play a role in the production of wear particles from the implant and microparticles of bone tissues and bone cement. Fibroproliferative process may be considered as an immune response to the implanted foreign material.

Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion.

Whole cell patch recordings were obtained from medium diameter (35-45 microm) dorsal root ganglion neurons. Using electrophysiological parameters, we were able to subclassify acutely dissociated dorsal root ganglion cells into three uniform (types 5, 6 and 9) and one mixed class (type 8) of neurons. All subtypes (types 5, 6, 8 and 9) had broad action potentials (7.0+/-0.2, 5.2+/-0.4, 7.3+/-0.5 and 6.0+/-0.4 ms) and exceptionally long afterhyperpolarizations (112+/-9, 178+/-19, 124+/-31 and 204+/-33 ms). Long afterhyperpolarizations have been linked to mechanically insensitive (silent) nociceptors by other laboratories [Djouhri et al., J. Physiol. 513 (1998) 857-872]. Chemosensitivity varied among cell classes. Cell types 5, 8 and 9 were capsaicin sensitive (45+/-13, 87+/-30 and 28+/-13 pA/pF; 5 microM) groups, while the type 6 cell was capsaicin insensitive. All cell types expressed ASIC-like (acid sensing ion channel) amiloride sensitive, proton-activated currents with a threshold of pH 6.8 and a peak near pH 5.0. All medium sized cells were sensitive to ATP (50 microM) and exhibited the 'mixed' form of ATP-gated current [Burgard et al., J. Neurophysiol. 82 (1999) 1590-1598; Grubb and Evans, Eur. J. Neurosci. 11 (1999) 149-154]. Immunohistochemistry performed on individual cells indicated the expression of both P2X(1) and P2X(3) subunits. Electrophysiologically defined classes were histochemically uniform. All types were examined for the presence of substance P (SP), calcitonin gene related peptide (CGRP) and binding of isolectin B4 (IB4). All subtypes expressed CGRP immunoreactivity. Types 5 and 8 co-expressed SP and CGRP immunoreactivity and also bound IB4. Subtypes 6 and 9 were positive for neurofilament m. It is likely that these cells represent major classes of myelinated and unmyelinated peptide expressing nociceptors.

Co-expression of P2X receptor subunits on rat nodose neurons that bind the isolectin GS-I-B4.

Triple fluorescent histochemistry was used to describe the types of overlap in visceral sensory neurons (nodose ganglion) for the labeling of the isolectin B4 from Griffonia simplicifolia type one (GS-I-B4) and their immunoreactivity (IR) for two of the ATP receptor subunits (P2X1/3 or P2X2/3). The vast majority of nodose neurons expressed GS-I-B4-binding and most of these displayed P2X receptor IR. Most of the P2X-IR was co-expressed on these individual nodose neurons (P2X1/P2X3 or P2X2/P2X3). A very small subpopulation of neurons that were GS-I-B4 negative but P2X positive displayed a very high relative intensity of P2X3-IR. The functional role that these expression patterns play in visceral sensory processing is currently unclear.

Diet restriction in rat toxicity studies: automated gravimetric dispensing equipment for allocating daily rations of powdered rodent diet into pouches and 7-day feeders.

Survival of ad libitum-fed rats has declined in the 2-year carcinogenicity bioassay. Restriction of the number of calories, without compromise of the overall nutrition offered to animals including rats, results in increased lifespan of animals. Diet restriction in rats is best achieved through offering of rations of feed daily instead of weekly, as is routinely done in ad libitum studies. The objective of this project was to develop an accurate and precise method of dispensing daily rations of feed. A gravimetric vibratory-type dispenser was used to dispense target weights of 15 and 20 g of powdered certified rodent diet into either labeled pouches or seven-well carousel feeders. The tolerance of the dispenser was the target weight +/- 3%. The amount of food offered to the diet-restricted rats was approximately 25% lower than that consumed by rats offered diet ad libitum. After 2 years, male rats offered 20 g and female rats offered 15 g of powdered rodent diet daily had remarkably lower body weights than did animals offered the diet ad libitum. Generally, the rats ate the entire ration of food offered to them each day. Survival of the diet-restricted rats was 70% to 82% at the end of a 2-year study. This investigation demonstrates that modest reduction of food intake, resulting in increased survival of Sprague Dawley rats in 2-year carcinogenicity bioassays, can be achieved reliably and efficiently through use of an accurate and precise automated method of dispensing powdered diet for use in multiple rat studies. In addition, this method of food dispensing provides a practical way to administer test compound in the diet under the conditions of diet restriction.

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types.

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.

Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents.

We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

P2X1 receptor subunit immunoreactivity and ATP-evoked fast currents in adult rat dorsal root ganglion neurons.

Triple fluorescent staining for P2X1 and P2X3 subunits and isolectin I-B4 (IB4) were performed on acutely dissociated rat DRG neurons. Immunoreactivity for P2X1 and P2X3 subunits was present separately or together in DRG neurons. P2X1 immunoreactivity was present in both IB4-positive and IB4-negative cells. When combining patch-clamp recordings with immunostaining for the P2X1 and P2X3 subunits on single recorded cells, ATP-evoked fast currents were shown to be present on DRG neurons that have immunoreactivity for the P2X3 subunit only, the P2X1 subunit only, or both P2X1 and P2X3 subunits. These results raised a possibility that, in addition to the P2X3 receptor subunit, the P2X1 subunit may also contribute to functional P2X receptors with fast kinetics in DRG neurons.

Distribution patterns of different P2x receptor phenotypes in acutely dissociated dorsal root ganglion neurons of adult rats.

P2x receptors may be used to detect ATP release from tissues during physiological and pathological conditions. We used whole-cell patch clamp recordings to study the expression of P2x receptor phenotypes, their distribution patterns, and their sensitivity to alphabetamATP and suramin in dorsal root ganglion (DRG) neurons acutely dissociated from adult rats. Based on the onset and decay rates of 10 microM ATP-evoked currents, we showed three types of P2x currents: fast, slow, and mixed. Each of these P2x receptor phenotypes had a distinct distribution pattern among DRG neurons. The fast P2x currents were predominantly expressed in small-diameter, isolectin-B4 (IB4)-positive, and capsaicin-sensitive DRG neurons. The slow P2x currents were expressed in both small and medium DRG neurons, and about half of them were IB4 positive. The mixed P2x currents were also expressed in both small and medium-sized DRG neurons, and most of these neurons were IB4-positive neurons. The slow and mixed P2x current groups had both capsaicin-sensitive and -insensitive DRG neurons. All phenotypes revealed with 10 microM ATP could be inhibited by 30 microM suramin. All DRG neurons with fast or mixed P2x currents were also sensitive to 10 microM alphabetamATP, and alphabetamATP evoked currents similar to those induced by ATP. The group expressing slow P2x currents could be further divided into alphabetamATP-sensitive and -insensitive groups. Thus, the relationships among P2x receptor phenotypes, cell sizes, IB4 positivity, and capsaicin sensitivity are more complicated than previously thought, and different P2x receptors may be involved in both nociceptive and non-nociceptive functions.

Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase.

Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g. (CAG)(16)(CTG)(4)), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 degrees C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG approximately GAC > CTG. Loops of GTC compared with CTG melt 9 degrees C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation.

Anodally focused polarization of peripheral nerve allows discrimination of myelinated and unmyelinated fiber input to brainstem nuclei.

We investigated the ability of a novel direct current (DC) polarization technique to block selectively the conduction in peripheral myelinated nerve fibers and allowing propagation in only unmyelinated fibers. In anesthetized adult rats, distal branches of the sciatic nerve (caudal cutaneous sural and tibial nerves) were exposed for electrical stimulation of A- and C-fibers. Two specially fabricated trough electrodes of different size and surface area were placed onto the sciatic nerve. Through these proximal electrodes a controlled ramped DC was timed to coincide with the arrival of A- and C-fiber action potentials, evoked electrically at the distal nerves or naturally from the foot or ankle, with the intent of blocking propagation in A-fibers while allowing C-fiber throughput. Neuronal recordings were made both peripherally (proximal sciatic nerve fascicles or L5 dorsal roots) and centrally (single cells in the nucleus gracilis or nucleus reticularis gigantocellularis). The DC polarization was shown to block conduction in myelinated A-fibers effectively, while allowing conduction in the unmyelinated C-fibers, without activation of fibers via the DC polarization itself. This was dependent upon the following factors: electrode polarity, onset rate of polarization, peak amplitude of polarization, distance between polarizing electrodes, size difference between polarizing electrodes, and gross nerve size. These experiments demonstrate that anodally focused DC polarization, applied utilizing two trough electrodes of different sizes, is capable of effectively, reversibly, and reproducibly blocking conduction in myelinated A-fibers evoked either electrically or naturally, while still allowing conduction to occur in the unmyelinated C-fiber population. In the context of experimental usage, we have demonstrated blocking of low-threshold A-fiber, but not C-fiber, mediated inputs to the caudal brainstem. This technique should find wide application in studies involving the processing of information conveyed centrally by the unmyelinated C-fiber afferent population, including discriminating afferent responses to peripheral stimuli, the role of C-fiber input in reflex activity, and the plasticity following injury or other manipulations.

Analysis of strand slippage in DNA polymerase expansions of CAG/CTG triplet repeats associated with neurodegenerative disease.

Lengthy expansions of trinucleotide repeats are found in DNA of patients suffering severe neurodegenerative age-related diseases. Using a synthetic self-priming DNA, containing CAG and CTG repeats implicated in Huntington's disease and several other neurological disorders, we measure the equilibrium distribution of hairpin folding and generate triplet repeat expansions by polymerase-catalyzed extensions of the hairpin folds. Expansions occur by slippage in steps of two CAG triplets when the self-priming sequence (CTG)16(CAG)4 is extended with proofreading-defective Klenow fragment (KF exo-) from Escherichia coli DNA polymerase I. Slippage by two triplets is 20 times more frequent than by one triplet, in accordance with our finding that hairpin loops with even numbers of triplets are 1-2 kcal/mol more stable than their odd-numbered counterparts. By measuring triplet repeat expansions as they evolve over time, individual rate constants for expansion from 4 to 18 CAG repeats are obtained. An empirical expression is derived from the data, enabling the prediction of slippage rates from the ratio of hairpin CTG/CTG interactions to CAG/CTG interactions. Slippage is initiated internally in the hairpin folds in preference to melting inward from the 3' terminus. The same triplet expansions are obtained using proofreading-proficient KF exo+, provided 10-100-fold higher dNTP concentrations are present to counteract the effect of 3'-exonucleolytic proofreading.

Cardiovascular effects after inhalation of large doses of albuterol dry powder in rats, monkeys, and dogs: a species comparison.

Albuterol is a quickly acting beta-2 adrenergic agonist bronchodilator widely used by asthmatics. Because recent case-control studies have suggested a relationship between the increase in mortality of asthmatics over the past decade and the use of beta 2-adrenergic agonists in the control of asthma, concern has developed regarding the potential cardiotoxicity of beta 2-specific adrenergic agonists, including albuterol. The aim of this investigation was to assess the potential for cardiotoxicity of inhaled albuterol dry powder in rats, monkeys, and dogs. All species were exposed to an aerosol of albuterol 1 h per day, 7 days per week, for at least 2 weeks. Control groups were exposed to filtered conditioned air and handled in the same manner as the albuterol-exposed animals. Plasma concentrations of albuterol confirmed systemic exposure. The daily inhaled dose received by the animals was calculated based on measured respiratory minute volumes, published respiratory tract deposition data, as well as HPLC-determined particle size distribution data and aerosolized albuterol concentrations. Multiples of the maximum daily clinical dose (presentation of 15 micrograms/kg in a 70-kg human) were approximately 0.25- to 2500-fold in the rat, 9- to 100-fold in the monkey, and 0.5- to 90-fold in the dog. No findings attributed to albuterol were observed in the monkey. Tachycardia and transient hypokalemia occurred in rats at multiples of 1.5 times or greater of the maximum clinical dose. Absolute and relative heart weights increased in rats receiving multiples of 47 times or greater of the maximum human dose. In the absence of histopathologic findings, the increases in rat heart weights were considered a physiologic hypertrophic response to tachycardia. In dogs tachycardia and transient hypokalemia occurred at all doses tested. Slight to mild fibrosis in the papillary muscles of the left ventricle of the heart occurred in dogs at multiples > or = 19 times the clinical dose. The cardiovascular effects observed were consistent with the known pharmacologic action of beta 2-adrenergic agonists. Due to the lack of toxicologically relevant findings in rats and monkeys and the wide safety margin in dogs, the findings in this study do not suggest a cardiotoxicity risk in the human population after repeated exposures to clinical doses of albuterol currently used in the treatment of asthma.

Localization of unmyelinated axons in rat skin and mucocutaneous tissue utilizing the isolectin GS-I-B4.

The alpha-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GS-I-B4) labels CNS microglia and certain peripheral neurons, including a subpopulation of small dark, type B dorsal root ganglion cells, some post-ganglionic sympathetic axons, and nearly all peripheral gustatory axons. The innervation patterns of GS-I-B4 reactive sensory ganglion cells are unknown for many peripheral target tissues, including their probable primary target, the skin. The present study describes the distribution of GS-I-B4 reactive axons in hairy and glabrous hindpaw skin and in the glans penis of rats, using both single and double-labelling histochemical techniques. Neuronal processes were identified using (1) histochemistry with horseradish peroxidase conjugated GS-I-B4 or (2) immunohistochemistry against PGP 9.5 to identify all axons, and biotinylated lectin histochemistry with avidin-FITC to identify the subpopulation of GS-I-B4 reactive axons. GS-I-B4 strongly labelled unmyelinated cutaneous sensory afferents, as well as some sympathetic efferents and visceral afferents. lectin reactive axons were seen to innervate the upper hair shaft epidermis in hairy skin, and were abundant in the shallow dermis in hairy and glabrous skin and glans penis. Lectin reactive axons were also abundant in the lamina propria and distal urethral epithelium of the penis. These results provide new evidence for the cutaneous sensory role of GS-I-B4 reactive primary afferents, as well as evidence to support the contention that the lectin is a specific marker for a subpopulation of unmyelinated axons and not simply a marker for the myelination state of an axon.

Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases.

Expansions of trinucleotide repeats in DNA, a novel source of mutations associated with human disease, may arise by DNA replication slippage initiated by hairpin folding of primer or template strands containing such repeats. To evaluate the stability of single-strand folding by repeating triplets of DNA bases, thermal melting profiles of (CAG)10, (CTG)10, (GAC)10 and (GTC)10 strands are determined at low and physiological salt concentrations, and measurements of melting temperature and enthalpy change are made in each case. Comparisons are made to strands with three times as many repeats, (CAG)30 and (CTG)30. Evidence is presented for stable intrastrand folding by the CAG/CTG class of triplet repeats. Relative to the GAC/GTC class not associated with disease, the order of folding stability is found to be CTG > GAC approximately = CAG > GTC for 10 repeats. Surprisingly, the folds formed by 30 repeats of CTG or CAG have no higher melting temperature and are only 40% more stable in free energy than those formed by 10 repeats. This finding suggests that triplet expansions with higher repeat number may result from the formation of more folded structures with similar stability rather than fewer but longer folds of greater stability.

Enthalpy-entropy compensation in DNA melting thermodynamics.

We investigate enthalpy-entropy compensation for melting of nearest-neighbor doublets in DNA. Based on data for 10 normal doublets and for doublets containing a mispaired or analog base, the correlation of delta Szero with delta Hzero follows a rectangular hyperbola. Doublet melting temperature relates linearly to delta Hzero by Tm = T(o) + delta Hzero/a, where T(o) = 273 K and a = 80 cal/mol-K. Thus Tm is proportional to delta Hzero + aTo rather than to delta Hzero alone as previously thought by assuming delta Szero to be constant. The term aTo = 21.8 kcal/mol may reflect a constant enthalpy change in solvent accompanying the DNA enthalpy change for doublet melting and is roughly equivalent to breaking four H-bonds between water molecules for each melted doublet. The solvent entropy change (aTo/Tm) declines with increasing Tm, while the DNA entropy change (delta Hzero/Tm) rises, so the combined DNA + solvent entropy change stays constant at 80 cal/K/mol of doublet. If such constancy in DNA + solvent entropy changes also holds for enzyme clefts as "solvent," then free energy differences for competing correct and incorrect base pairs in polymerase clefts may be as large as enthalpy differences and possibly sufficient to account for DNA polymerase accuracy. The hyperbolic relationship between delta Szero and delta Hzero observed in 1 M salt can be used to evaluate delta Hzero and delta Szero from Tm at lower, physiologically relevant, salt concentrations.

Does physiologic gastroesophageal reflux affect heart rate or rhythm?

An acid-induced, cholinergic esophagocardiac reflex has been observed in humans. Decrements of heart rate can be induced by direct intraesophageal acid infusion. To ascertain whether this reflex occurs during physiologic reflux and whether stimulation of this reflex might precipitate dysrhythmias, a prospective study was performed. Twenty consecutive patients referred for 24-h ambulatory intraesophageal pH monitoring underwent simultaneous 24-h cardiac holter monitoring. Analyses were performed only on gastroesophageal reflux episodes which resulted in esophageal acidification to pH < 4 for 60 sec or more. Evaluable cardiac holter variables included premature ventricular contractions (PVCs), premature atrial contractions (PACs), tachycardia (heart rate, > 110/min), and bradycardia (heart rate, < 50/min). Measurements were made for 60 sec before and after onset of esophageal acidification. No relationship was noted between physiologic episodes of gastroesophageal reflux and PVCs (p = 0.29), PACs (p = 0.12), tachycardia (p = 0.33), or bradycardia (p = 0.78). No statistically significant correlations were noted between total 24-h acid exposure (minutes/24h) and mean heart rate (p = 0.07), number of PVCs (p = 0.41), and number of PACs (p = 0.39). Analyses of reflux episodes lasting more than 5 min with intraesophageal pH < 2 also failed to show changes in pulse rate (p = 0.22). Physiologic gastroesophageal reflux does not induce changes in heart rate or rhythm in humans. It is possible that esophagocardiac reflexes noted during intraesophageal acid infusion are related to lower pH values or to other factors such as osmolarity, temperature, or site-specific receptors.

Biochemical basis of DNA replication fidelity.

DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using polyacrylamide gel electrophoresis (PAGE) to resolve 32P-labeled primer molecules extended by polymerase. We describe how integrated gel band intensities can be used to obtain site-specific velocities for addition of correct and incorrect nucleotides, extending mismatched compared to correctly matched primer termini and measuring polymerase dissociation rates and equilibrium DNA binding constants. The analysis is based on steady-state "single completed hit conditions", where polymerases encounter many DNA molecules but where each DNA encounters an enzyme at most once. Specific topics addressed include nucleotide misinsertion, mismatch extension, exonucleolytic proofreading, single nucleotide discrimination using PCR, promiscuous mismatch extension by HIV-1 and AMV reverse transcriptases, sequence context effects on fidelity and polymerase dissociation, structural and kinetic properties of mispairs relating to fidelity, error avoidance mechanisms, kinetics of copying template lesions, the "A-rule" for insertion at abasic template lesions, an interesting exception to the "A-rule", thermodynamic and kinetic determinants of base pair discrimination by polymerases.

Myeloperoxidase-enhanced formation of (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene-DNA adducts in lung tissue in vitro: a role of pulmonary inflammation in the bioactivation of a procarcinogen.

Several studies have indicated a correlation between the presence of inflammation and the development of cancer. The aim of our study was to determine if pulmonary neutrophils could transform the proximate respiratory carcinogen (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol), to an ultimate carcinogenic metabolite via myeloperoxidase (MPO). To test this hypothesis, virus-free male DBA/2 mice were exposed by inhalation to the Gram-negative bacteria Proteus mirabilis for 1 h. For various time points post-exposure, bronchoalveolar lavage (BAL) was performed to determine total and differential cell counts, cellular MPO activity and production of superoxide. Twelve hours after the exposure, cellular activity of MPO as well as percentage and total number of polymorphonuclear leukocytes peaked and declined thereafter. At this same time point, cells from BAL exhibited increased release of superoxide, as measured by reduction of cytochrome c, after addition of soluble or particulate stimuli, 12-O-tetradecanoylphorbol-13-acetate (TPA) or opsonized zymosan respectively. These cells also elicited biotransformation of B[a]P-7,8-diol as evidenced by enhanced B[a]P-7,8-diol-derived chemiluminescence, tetraol formation and covalently bound adduct formation to exogenous DNA upon addition of TPA or opsonized zymosan. Moreover, the cell-free BAL fluid of infected mice contained substantial MPO activity in comparison to that of uninfected animals. Also, MPO enhanced the binding of B[a]P-7,8-diol to lung DNA in vitro. Unlike previous work emphasizing the potential roles of oxygen free radicals in tumor promotion, our results indicate a role of neutrophilic MPO in the initiation of carcinogenesis.

Enhanced lipid peroxidation in lung lavage of rats after inhalation of asbestos.

Exposure of phagocytic cells to asbestos in vitro results in an augmented production of reactive oxygen metabolites and increased peroxidation of lipids. The aim of this investigation was to assess the extent of lipid peroxidation both in cells and fluid obtained from bronchoalveolar lavage (BAL), and in lungs of rats exposed to crocidolite asbestos or titanium dioxide (TiO2), a nonfibrous particulate control. In comparison to sham and TiO2-exposed rats, the BAL fluid and cells of crocidolite-exposed animals contained significantly elevated levels of malondialdehyde (MDA), a breakdown product of lipid peroxidation detected using high-pressure liquid chromatography (HPLC). In contrast, no significant differences in MDA were detected in lavaged lung tissue from these animals. Inhalation of crocidolite caused an early inflammatory response characterized by elevated numbers of polymorphonuclear leukocytes and lymphocytes, as well as enhanced total protein in BAL. Pulmonary fibrosis and increased lung hydroxyproline also were observed after 20 days of exposure. Exposure to TiO2 did not cause inflammation, pulmonary fibrosis, or elevated amounts of hydroxyproline in the lung. Our results show that exposure to the fibrogenic and inflammatory mineral, crocidolite, results in an enhanced lipid peroxidation in BAL cells and fluid not observed after inhalation of the particulate TiO2. These novel observations suggest that MDA in BAL may be useful as a biomarker of exposure to inhaled asbestos or other oxidants.

Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis.

Several in vitro studies suggest the involvement of active oxygen metabolites in cell damage caused by asbestos. To determine if lung injury, inflammation, and asbestosis could be inhibited in vivo in a rapid-onset, inhalation model of disease, a novel method of chronic administration of antioxidant enzymes was developed. In brief, Fischer 344 rats were treated with polyethylene glycol-conjugated (PEG-) superoxide dismutase or catalase in osmotic pumps over a 10-day (5 days/wk for 2 wk) or 20-day (5 days/wk for 2 wk) period of exposure to crocidolite asbestos. Control rats included sham-exposed animals and those exposed to asbestos but receiving chemically inactivated enzymes. After 10 days of exposure to asbestos, lactic dehydrogenase (LDH), alkaline phosphatase, and total protein in bronchoalveolar lavage (BAL) were measured in one group of rats. Total and differnetial cell counts in BAL also were assessed. After 20 days of exposure, lungs of an additional group of rats were evaluated by histopathology and by measurement of hydroxyproline. Asbestos-associated elevations in LDH, protein, and total cell numbers in BAL were reduced in rats receiving PEG-catalase. Decreases in numbers of alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes occurred in these animals. Exposure to asbestos for 20 days caused significant increases in both the amount of hydroxyproline in lung and the severity and extent of fibrotic lesions as determined by histopathology. These indicators of asbestosis were inhibited in a dosage-dependent fashion in rats receiving PEG-catalase. Use of inactivated PEG-catalase failed to boost serum levels of catalase and did not inhibit asbestos-induced elevation of hydroxyproline in lung.(ABSTRACT TRUNCATED AT 250 WORDS)