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Inhibition of biofilm formation by monoclonal antibodies against Staphylococcus epidermidis RP62A accumulation-associated protein.

Staphylococcus epidermidis expresses a 140-kDa cell wall-bound protein accumulation-associated protein (AAP) to adhere to and accumulate as a biofilm on a surface. Potentially blocking AAP with a monoclonal antibody (MAb) could reduce or eliminate S. epidermidis bacterial colonization of biomedical devices. Here, we report on our efforts to (i) isolate AAP, (ii) generate MAbs against AAP, and (iii) determine the efficacy of MAbs to inhibit S. epidermidis biofilm formation. An M7 S. epidermidis mutant, reportedly deficient in AAP expression, was used as a negative control. Postinoculation murine sera, containing polyclonal antibodies against AAP, were able to reduce S. epidermidis biofilm formation by 54%. Select MAbs against AAP were able to reduce S. epidermidis by no more than 66%. Two MAb mixtures, 12C6/12A1 and 3C1/12A1, reduced S. epidermidis accumulation up to 79 and 87%, respectively, significantly more than individual MAbs. Contrary to a previous report, biofilm-deficient S. epidermidis mutant M7 expressed a 200-kDa protein on its cell wall that specifically bound AAP MAbs. Peptide characterization of this M7 protein by microcapillary reversed-phase high-pressure liquid chromatography-nanoelectrospray tandem mass spectrometry resulted in 53% homology with AAP. Ongoing studies will elucidate the dynamic expression of AAP and the M7 200-kDa protein in order to define their roles in biofilm formation.

Amino Acid Sequence↗

Gamma-glutamyltransferase is a reliable marker for tubular effects of contrast media.

The aim of this study was to evaluate the usefulness of the measurement of urinary excretion of the brush-border enzyme gamma glutamyl-transferase (GGT), in comparison with that of alanine aminopeptidase (AAP), as a marker for tubular toxicity due to contrast media (CM). Urinary activities of AAP and GGT were measured prior to the administration of CM and 1, 3 and 5 days after in forty-nine adult renal patients undergoing a radiological examination with intravascular administration of CM. The behavior of GGT was similar to that of AAP. In fact, urinary activities of both AAP and GGT increased greatly after CM. This effect was maximal on the 1st day and statistically significant for both enzymes. Furthermore, on the 1st day a relevant increase of enzyme activity (at least +50% over the basal value) was observed in the same number of patients (67%) for AAP and GGT. The concordance between GGT and AAP variations was high and statistically significant. Finally, different variables (osmolarity, dose of CM, and baseline renal function of the patients) had a similar effect on urinary excretion of AAP and GGT. The repeatability of duplicated determinations of GGT resulted better than that of AAP. In conclusion, the good concordance of the results of GGT with those of AAP justifies the use of GGT as a marker for tubular effects due to CM. Furthermore, the measurement of GGT has a better repeatability than that of AAP.

Adult↗

Alanine aminopeptidase in serum: biological variations and reference limits.

We studied the effect of various factors on the biological variation of alanine aminopeptidase (AAP, EC 3.4.11.2) in serum in a population of 2178 apparently healthy subjects and in subjects taking specific drugs. To measure AAP, we used an automated assay, with alanyl-4-nitroanilide as substrate. AAP activity concentrations were significantly higher in males than in females in all age groups between 10 and 55 years. The highest mean AAP values were found for children between 10 and 14 years. In male heavy smokers, AAP values were 8% higher than in moderate or nonsmokers (P less than 0.001); in females, this effect was less pronounced. AAP concentrations were higher in subjects consuming over 44 g of ethanol a day than in those consuming less (P less than 0.01 for men, P less than 0.05 for women). The use of oral contraceptives increased AAP values by 12% (P less than 0.001). Hypolipidemic drugs caused lower values for AAP in men (P less than 0.001). Subjects showing induction of gamma-glutamyltransferase (EC 2.3.2.2) by anticonvulsant drugs had also higher AAP activities (by 23%). Taking these data into account, we established reference limits for AAP in serum.

Adolescent↗

Residues on Adeno-associated Virus Capsid Lumen Dictate Interactions and Compatibility with the Assembly-Activating Protein.

The adeno-associated virus (AAV) serves as a broadly used vector system for in vivo gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly.IMPORTANCE Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

Amino Acid Sequence↗

Detection of early visual field loss in glaucoma using frequency-doubling perimetry and short-wavelength automated perimetry.

OBJECTIVE: To investigate whether frequency-doubling perimetry (FDP) predicts future visual field loss with achromatic automated perimetry (AAP), just as it may be predicted with short-wavelength automated perimetry (SWAP). METHODS: We recruited 62 patients selectively from an urban glaucoma practice. At the commencement of the study, each patient had ocular hypertension with normal visual fields on AAP. Baseline SWAP and FDP were performed to determine whether underlying earlier visual field loss was present. Patients were then followed up prospectively for 3 years with annual AAP, SWAP, and FDP. Main Outcome Measure The development of visual field loss on AAP. RESULTS: Nine subjects had abnormal SWAP findings and 10 had abnormal FDP findings. At the conclusion of the study, field loss on AAP developed in 5, all of whom had preexisting abnormal SWAP and FDP results. No AAP visual field loss developed in patients with a normal SWAP or FDP. The rate of development of visual field loss on AAP was therefore significantly greater for those with abnormal SWAP (chi21 = 40.83; P<.001) and abnormal FDP findings (chi21 = 32.76; P<.001) than for those with normal SWAP and FDP findings. CONCLUSION: In the same way that SWAP may predict AAP visual field loss, FDP may also detect field loss earlier than AAP.

Female↗

Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice.

The mechanism of liver cell injury induced by an overdose of the analgesic acetaminophen (AAP) remains controversial. Recently, it was hypothesized that a significant number of hepatocytes die by apoptosis. Since caspases have been implicated as critical signal and effector proteases in apoptosis, we investigated their potential role in the pathophysiology of AAP-induced liver injury. Male C3Heb/FeJ mice were fasted overnight and then treated with 500 mg/kg AAP. Liver injury became apparent at 4 h and was more severe at 6 h (plasma ALT activities: 4110 +/- 320 U/liter; centrilobular necrosis). DNA fragmentation increased parallel to the increase of plasma ALT values. At 6 h there was a 420% increase of DNA fragmentation and a 74-fold increase of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells located predominantly around central veins. However, the activity of the proapoptotic caspase-3 was not increased at any time after AAP. In contrast, injection of the anti-Fas antibody Jo-2 (positive control) caused a 28-fold increase of caspase-3 activity and severe DNA fragmentation before significant ALT release. Treatment with the caspase inhibitor ZVAD-CHF2 had no effect on AAP toxicity but completely prevented Jo-mediated apoptosis. In contrast, Jo-induced caspase activation and apoptosis could be inhibited by AAP treatment in a time- and dose-dependent manner. We conclude that AAP-induced DNA fragmentation does not involve caspases, suggesting a direct activation of endonucleases through elevated Ca2+ levels. In addition, electrophilic metabolites of AAP may inactivate caspases or their activation pathway. This indicates that AAP metabolism has the potential to inhibit signal transduction mechanisms of receptor-mediated apoptosis.

Acetaminophen↗

Isolation, identification, and accumulation of 2-acetamidophenol in liquid cultures of the wheat take-all biocontrol agent Pseudomonas fluorescens 2-79.

Pseudomonas fluorescens strain 2-79 (NRRL B-15132) is a classic biological control agent known to produce phenazine-l-carboxylic acid (PCA) as its primary means of suppressing take-all disease of wheat. In addition to PCA, an unknown metabolite was discovered in a liquid culture used to produce the biocontrol agent. The objective of the current study was to isolate, identify, and evaluate the accumulation of this compound in production cultures. Upon centrifugal fractionation of a production culture, thin-layer chromatography (TLC) analyses of extracts of the cells and cell-free supernatant indicated the compound to be primarily in the supernatant. Purified compound was obtained by extraction of culture supernatant, followed by flash chromatography of the extract and preparative TLC. The 'H and 13C nuclear magnetic resonance and electron impact mass spectra indicated the compound to be 2-acetamidophenol (AAP). Measured by reversed-phase HPLC, the accumulations of AAP and PCA in cultures of strain 2-79 reached 0.05 g/l and 1 g/l, respectively. The accumulations of AAP and PCA in liquid cultures were linearly correlated (P < 0.001), as shown by studies of cultures stimulated to yield varying levels of PCA by controlling levels of oxygen transfer, pH, and growth medium composition. In this study, oxygen limitation, a defined amino-acid-free medium, and neutral pH stimulated maximal production of both AAP and PCA. Furthermore, a transposon mutant of 2-79 [2A40 2-79 (phz-)] unable to produce PCA did not accumulate AAP. These findings indicate that AAP and PCA are likely to share a common segment of biosynthetic pathway. This is the first report of AAP production by a strain of P. fluorescens. Possible routes of AAP production are discussed relative to current knowledge of the phenazine biosynthetic pathway of strain 2-79. The pertinence of AAP to the design of commercial seed inoculants of phenazine-producing bacteria for controlling wheat take-all is also considered.

Acetaminophen↗

Protective effects of ginkgo biloba against acetaminophen-induced toxicity in mice.

BACKGROUND: The analgesic acetaminophen (AAP) causes a potentially fatal, hepatic centrilobular necrosis when taken in overdose. It was reported that these toxic effects of AAP are due to oxidative reactions that take place during its metabolism. OBJECTIVE: In this study, we aimed to investigate the possible beneficial effect of Ginkgo biloba (EGb), an antioxidant agent, against AAP toxicity in mice. METHODS: Balb/c mice were injected i.p. with: (1) vehicle, control (C) group; (2) a single dose of 50 mg/kg Ginkgo biloba extract, EGb group; (3) a single dose of 900 mg/kg i.p. acetaminophen, AAP group, and (4) EGb, in a dose of 50 mg/kg after AAP injection, AAP + EGb group. Serum ALT, AST, and tumor necrosis factor-alpha (TNF-alpha) levels in blood and glutathione (GSH), malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and collagen contents in liver tissues were measured. Formation of reactive oxygen species in hepatic tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lusigenin probe. Tissues were also examined microscopically. RESULTS: ALT, AST levels, and TNF-alpha were increased significantly (p < 0.001) after AAP treatment, and reduced with EGb. Acetaminophen caused a significant (p < 0.05-0.001) decrease in GSH levels while MDA levels and MPO activity were increased (p < 0.001) in liver tissues. These changes were reversed by EGb treatment. Furthermore, luminol and lusigenin CL levels in the AAP group increased dramatically compared to control and reduced by EGb treatment (p < 0.01). CONCLUSION: Our results implicate that AAP causes oxidative damage in hepatic tissues and Ginkgo biloba extract, by its antioxidant effects protects the tissues. Therefore, its therapeutic role as a "tissue injury-limiting agent" must be further elucidated in drug-induced oxidative damage.

Acetaminophen↗

Investigation of possible mechanisms of hepatic swelling and necrosis caused by acetaminophen in mice.

Vascular congestion and liver swelling have long been recognized as features of the hepatotoxic effects of acetaminophen (AAP) in mice and rats and have been proposed as contributing factors to the eventual extent of necrosis produced. Neutrophil accumulation in the hepatic microcirculation has been proposed as being responsible for the blockage of hepatic blood flow and thereby the expansion of the region of damage. We therefore determined in mice the effects of hepatotoxic doses of AAP on the messenger RNA for intercellular adhesion molecule-1 (ICAM-1), which is a critical determinant of neutrophil adhesion, activation and ultimately of neutrophil-mediated tissue injury. Hepatotoxic doses of AAP did not upregulate ICAM-1 messenger RNA. However, doses of bacterial lipopolysaccharide (LPS) did cause a rapid and dramatic increase in ICAM-1 message, which was accompanied by a much greater hepatic accumulation of neutrophils, but which led to only scattered single cell necrosis. In addition, we investigated the effects of pentoxifylline (PTX) on AAP-induced vascular congestion and on hepatic necrosis as evaluated histologically and by measurement of plasma transaminase activities. Although PTX has been shown to increase blood cell deformability and improve vascular perfusion in a number of animal models of restricted blood flow, and is used in humans for the treatment of intermittent claudication, we found no decrease in AAP-induced hepatic swelling or in AAP-induced necrosis in response to PTX. With some dosing regimens, PTX-treated animals proved to be slightly more susceptible to AAP, which may be related to the reported potentiation of the cytotoxicities of a number of alkylating anti-cancer drugs by PTX and other methylxanthines. We conclude from these studies that upregulation of ICAM-1 and subsequent adhesion and vascular plugging by neutrophils are not significant determinants of AAP-induced liver swelling and necrosis and that whatever hemorheological advantages PTX might offer in AAP-induced hepatic damage appear to be overshadowed by effects that potentiate the toxic responses.

Acetaminophen↗

Acetaminophen hepatotoxicity and mechanisms of its protection by N-acetylcysteine: a study of Hep3B cells.

Acetaminophen (AAP) hepatotoxicity, resulting in centrilobular necrosis, is frequently encountered following suicidal attempts, especially by adolescents, but also after its excessive use in infants. The subcellular and molecular sequences leading to hepatocellular cell death are not yet clear. We therefore investigated AAP hepatotoxicity by using cultured hepatoma-derived cells (Hep3B) exposed to AAP and N-acetylcysteine (NAC), used as a protective agent. Specifically, we studied the role of apoptosis and oxidative damage as putative mechanisms of AAP-associated cytotoxicity. Hep3B cells were exposed to AAP (5-25 mM) and NAC (5 mM) for different time periods. Cell viability was assessed by the Alamar Blue Reduction Test and LDH. Oxidative damage was evaluated by measuring reactive oxygen species (ROS) and glutathione. AAP-induced apoptosis was investigated by flow cytometry and transmission electron microscopy. We found that: 1. In Hep3B cells, AAP causes a time- and concentration-dependent cytotoxic effect, leading to oxidative stress, mitochondrial dysfunction, alterations of membrane permeability and apoptosis; 2. In the course of AAP cytotoxicity, the generation of ROS appears as an early event which precedes decrease of viability, LDH leakage, glutathione depletion and apoptosis; 3. NAC protects Hep3B cells from AAP-induced oxidative injury, but does not prevent apoptosis.

Acetaminophen↗

Chronic renal tubular effects in relation to urine cadmium levels.

In this study the urine activities of two brush border membrane enzymes, alanine aminopeptidase (AAP) and gamma-glutamyltranspeptidase (GGT), and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAG) were measured in men exposed to cadmium to investigate chronic renal toxicity. The subjects consist of a group with urine cadmium levels less than 2.0 micrograms/l and a group with higher cadmium levels (urine cadmium greater than or equal to 2.0 micrograms/l) with past or present occupational exposure to cadmium. The mean NAG value corrected for creatinine in the higher cadmium group (2.95 U/g creatinine) is significantly different from the mean low cadmium group value (0.92 U/g creatinine, p = 0.0083). Likewise, AAP in the higher group (13.83 U/g creatinine) is significantly different from that of the low group (6.58 U/g creatinine; p = 0.0018). NAG and AAP also give significant correlations with cadmium levels in urine (NAG: r = 0.51; p = 0.0001; AAP: r = 0.56; p = 0.0001). There is no similar statistically significant difference in GGT means in the two groups. In contrast to cadmium correlations, blood lead does not correlate with NAG, AAP, or GGT. A dose-response relationship was found between NAG and cadmium and between AAP and cadmium. The analysis of this relationship gives estimates of a 10% chance of observing an elevated NAG value at a cadmium level of 6.3 micrograms/g creatinine (8.0 micrograms/l) and a 10% chance of observing an elevated AAP at a cadmium level of 5.0 micrograms/g creatinine (3.4 micrograms/l). These data indicate elevations of NAG and AAP at urine cadmium levels below the level of 10 micrograms/g creatinine recommended as an upper limit by the 1980 World Health Organization Study Group. Since elevated levels of NAG and AAP in cadmium-exposed workers may reflect chronic renal tubular nephrotoxicity, these findings indicate that cadmium levels below 10 micrograms/g creatinine may be accompanied by subclinical tubular dysfunction, and that WHO guidelines should be interpreted cautiously, particularly with reference to workers who are no longer exposed and may have had higher cadmium body burdens in the past.

Acetylglucosaminidase↗

Protective effect of moutan cortex extract on acetaminophen-induced cytotoxicity in human Chang liver cells.

The aim of this study was to investigate the effect of Moutan Cortex on acetaminophen (AAP)-induced toxicity in human Chang liver cells. Cells were incubated with AAP (0-30 mM) to evaluate the drug's ability to reduce cytoviability. For the cells treated with 10, 20 and 30 mM AAP, LDH leakage was 39.8%, 49.0% and 57.6%, respectively. Administration of Moutan Cortex reduced cytotoxicity in a dose-dependent manner. Glutathione (GSH) concentration in human liver cells decreased significantly after exposure to 20 (p<0.05) and 30 mM (p<0.01) AAP, and increased (p<0.05) if incubated with AAP and Moutan Cortex. The ability of AAP to inhibit mitochondrial function and its counteraction by Moutan Cortex was also evaluated. Moutan Cortex showed dose-dependent increases in MTT metabolism and ATP levels in AAP-treated cells. The DNA content of AAP-treated cells increased with the treatment of Moutan Cortex. These observations demonstrate that Moutan Cortex may significantly attenuate AAP-induced toxicity. It can be considered a cytoprotective agent in this in vitro model of drug toxicity.

Acetaminophen↗

Use of 2-acetamidophenanthrene and 2-acetamidofluorene in investigations of mechanisms of hepatocarcinogenesis.

Comparison studies have been undertaken on the hepatocarcinogen 2-acetamidofluorene (AAF) and its nonhepatocarcinogenic analog 2-acetamidophenanthrene (AAP). Previous studies have shown that amount of compound acutely and persistently bound to rat liver DNA is comparable for the two compounds following single injections into adult Fischer rats, but that AAP fails to initiate tumors in weanling Sprague-Dawley rats. In this work we show that the amount of bound adduct from AAF and AAP is also comparable after three weeks of feeding compound to weanling Sprague-Dawley male rats. Three of the adducts found in RNA of AAP-treated rats cochromatographed on Sephadex LH-20 with two adenosine adducts and one guanosine adduct prepared by reaction of the nucleosides with N-acetoxy-N-trifluoroacetyl-2-aminophenanthrene at neutrality. Because of the lack of initiating ability of AAP in liver, we have also investigated early biochemical alterations in liver after various regimens. Feeding of either AAF or AAP to male weanling rats, followed by three weeks of DDT feeding produced no alteration in either histochemically detected gamma-glutamyltranspeptidase or in ganglioside complement in total liver homogenate. Partial hepatectomy after feeding of either AAF or AAP resulted in the appearance of new fucoganglioside and alteration in the distribution of the major gangliosides. DDT feeding after partial hepatectomy resulted in foci of elevated gamma-glutamyltranspeptidase in AAF-fed rats but not in AAP-fed rats. These results support a previous proposal that AAP may initiate tumorigenesis in rat liver, but that the promoting regimens now in use lack the ability to cause further progression of the initiated cells. The data also suggests that ganglioside synthesis may be a more sensitive marker for early stages in carcinogenesis than are the various histochemical stains now in use.

2-Acetylaminofluorene↗

Oral rehydration, emergency physicians, and practice parameters: a national survey.

BACKGROUND/OBJECTIVE: Treatment of dehydrating gastroenteritis, a major cause of morbidity in children, remains controversial. Practice parameters issued by the American Academy of Pediatrics (AAP) recommend oral rehydration therapy (ORT) as the preferred treatment for losses from both mild and moderate dehydration. The objective of this study was to test the hypothesis that awareness of the AAP practice parameters would be associated with a higher rate of use of ORT among emergency physicians. METHODS: A national random survey of emergency physicians selected from the mailing list of the AAP Section on Emergency Medicine was conducted. Respondents were asked about level of awareness of AAP guidelines and preferred route of rehydration for scenarios representing mild (< or =5%), moderate (6%-9%), and severe (> or =10%) dehydration. Respondents who were very familiar with the AAP practice parameters were designated as the familiar group; those who were less familiar were designated as the unfamiliar group. Differences in proportions of dichotomous variables were tested by chi2 or Fisher exact test as appropriate. Univariate logistic regression modeling was used to determine the independent influence of awareness of AAP guidelines on the use of ORT. RESULTS: A total of 176 physicians responded (73% response rate). Most were board certified in 1 or more specialties: pediatrics (89%), pediatric emergency medicine (84%), and adult emergency medicine (63%). Median duration of practice was 16 years. Thirty-seven percent of respondents were very familiar with the AAP practice parameters. In contrast to the unfamiliar group, the familiar group was more likely to use ORT in scenarios of mild dehydration (81% vs 66%) and moderate dehydration (25% vs 10%). Logistic regression analysis confirmed that this familiar group overall was nearly 3 times more likely to use ORT (odds ratio: 2.85; 95% confidence interval: 2.46-3.24). CONCLUSIONS: This is the first national study to establish a relationship between awareness of the AAP practice parameters and physician practices in the treatment of dehydration. Emergency medicine physicians who were very familiar with the AAP parameters were significantly more likely to use ORT. These findings may have important implications for future efforts directed at increasing the rate of ORT use and for the evaluation of practice parameters in general.

Adult↗

Policy versus practice: comparison of prescribing therapy and durable medical equipment in medical and educational settings.

OBJECTIVE: The American Academy of Pediatrics (AAP) has promoted pediatrician involvement in the care of children with special health care needs (CSHCN), including the prescription and/or supervision of therapies and durable medical equipment (DME) for children in both medical and educational settings, such as schools and early intervention programs. Through this survey, we attempted to quantify objectively how pediatricians direct and coordinate therapy and DME for CSHCN and how these efforts correlate with AAP recommendations. METHODS: A survey was mailed to a random sample of 500 physicians listed in the AAP directory, resulting in a final sample of 217 responding physicians who indicated that they provide services to CSHCN. Results of the survey were reported as proportions, means with standard deviations, or medians with interquartile range. Comparisons of proportions among certain subgroups of interest were made using Fisher exact tests. RESULTS: The most recent AAP policy revision addressing the role of physicians in prescribing therapy services for children with motor disabilities appeared in Pediatrics 1996. It listed 6 key items that should be part of a therapy prescription: diagnosis, precautions, type, frequency, anticipated goals (educators may prefer the term "objectives"), and duration. The policy addressed and emphasized the need for what may be additional objectives, namely regular communication between all parties involved, ongoing supervision and reevaluation of the program and problem, and awareness of other community resources for possible referrals. Except for providing a diagnosis, the majority of surveyed pediatricians do not regularly comply with AAP policy recommendations on prescribing therapies and DME in medical and educational settings. Physicians who were trained before 1980 tend to follow AAP recommendations more closely than later graduates. Decreasing involvement of private outpatient pediatricians in coordinating and supervising CSHCN's care was noted. Furthermore, the majority is willing to defer decisions about treatment and goals to nonphysician health care providers (NPHCPs) and, in some cases, even equipment vendors. More than two thirds of the respondents indicated that they would sign a prescription for therapy without their previous initiation if it had been initiated by a therapist. Likewise, most respondents said that they would sign a wheelchair prescription sent to them by a therapist. Few expressed confidence in determining the appropriateness of leg brace (orthosis) prescriptions and arm/hand brace prescriptions. The majority of survey participants said that they give open-ended length of time (no limits under 1 year) on prescriptions for therapy services as part of school-based programs. However, patients' conditions and their therapeutic or equipment needs may change during the school year. Because open-ended prescriptions do not require periodic renewal, they do not provide opportunities for periodic feedback that helps to ensure that the pediatrician is kept abreast of the patient's status and progress. The majority of respondents indicated that they would see a patient before signing either a therapy or DME prescription if they had not seen that patient in the past year. A little more than half of survey respondents said that they would participate initially in recommending which professional services or therapies should be performed as part of early intervention programs most of the time, but one third said that they participated less than half the time and approximately 14% said that they never participated. A majority would require being involved before authorizing therapy services as part of a school-based program, but a substantial minority would provide retroactive authorization for services that they did not initiate themselves. More than three quarters of respondents would prefer to let the therapist or educator set the goals. Only 58% of pediatricians reported receiving a detailed progress report once or twice a year, and approximately one fifth received no reports on patients in school-based programs. A literature review suggested that there are different perceptions among physicians and educationally based service providers regarding the physician's role in initiating and supervising educationally based services and equipment, which may influence the extent of physician involvement. AAP and other professional organizations, such as the American Medical Association and the American Academy of Physical Medicine and Rehabilitation, as well as federal guidelines and third-party payers emphasize the important role of physicians in initiating, determining the medical necessity, and ordering of services as well as in ongoing patient treatment. If therapists through their states' scope of practice guidelines have autonomy of practice or if the school self-funds educationally based services, then there may be no issues regarding physician authorization. However, if a physician's authorization is required for reimbursement, then the physician's professional, legal, and practice guidelines come into play. Physicians should be conscientious about fulfilling their responsibilities in serving as the medical home and supervising and monitoring medical services for their patients in both community and educational settings. Failure to properly fulfill the responsibilities inherent in signing a prescription may bring adverse consequences for the patient as well subject the physician to legal liability if adverse events occur. CONCLUSIONS: Ideally, there should be a seamless continuity and cooperation among the environments of medicine, home, community, and education rather than separate and perhaps conflicting domains. All health care professionals and other service providers involved should be acknowledged as collaborative team members. Except for provision of the diagnosis, the majority of surveyed pediatricians do not comply with AAP policy recommendations on prescribing community/medical-based and educationally based services for CSHCN. Furthermore, the majority are willing to defer these decisions to other NPHCP. This raises issues regarding overall continuity of care versus care of the child in a variety of environments, the concept of the medical home, and legal risk as a result of failure to follow federal and state practice guidelines. Also, there seem to be different cultural perceptions among physicians and educationally based service providers regarding the physician's role in educationally based services. These cultural differences should be explored further to promote a greater collegial cooperation and understanding. Decreasing involvement of private outpatient pediatricians in coordinating and supervising CSHCN care and a trend toward greater deference to NPHCP since 1979 were noted. If the numerous policies and guidelines previously promoted by AAP have not had a significant impact on pediatrician practices in these fields, then other, more effective alternatives should be explored.

Child↗

Hepatoprotective effects of the water extract from adzuki bean hulls on acetaminophen-induced damage in rat liver.

We examined the hepatoprotective effect of water-extract from adzuki bean (Vigna angularis) hulls on acetaminophen (AAP)-induced damage in rat liver. F344/DuCrj rats of 8 weeks of age were fed diets without and with 0.5% AAP or besides it 5% adzuki extract (lyophilized) on a daily basis over a period of 4 wk. At that time, serum aspartate aminotransferase activity in only AAP-treated group was higher than in both control and AAP plus adzuki extract (AAPA)-treated groups, while hepatic glutathione content and hepatic glutathione reductase and catalase activities in the AAP-treated group were lower than in the control group in contrast to the reverse in the AAPA-treated group. Hepatic phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide concentrations were higher in the AAP-treated group than in the control group, and were lower in the AAPA-treated group than in the AAP-treated group. Hepatic glutathione peroxidase activity was higher in the AAP-treated group than in the control group, although there was no significant difference between both AAP- and AAPA-treated groups in this respect. These findings suggest that the adzuki extract will serve as a prophylactic against oxidative damage to the liver.

Acetaminophen↗

The effect of verapamil on the nephrotoxic potential of gentamicin as measured by urinary enzyme excretion in healthy volunteers.

The effects of verapamil on the nephrotoxic potential of multiple-dose gentamicin were studied in healthy adult male volunteers. Subjects received a gentamicin infusion every 8 hours for 19 doses. Gentamicin dosage was adjusted to maintain peak concentrations of 5.5 mg/L and trough concentrations of 0.5 mg/L. Verapamil was given as a sustained release preparation of 180 mg twice daily starting 2 days before the aminoglycoside, and continued for 4 days post-gentamicin therapy. Nephrotoxicity was assessed by measuring 24-hour urinary alanine aminopeptidase excretion (AAP). The urinary AAP results of six subjects given gentamicin with verapamil were compared with urinary AAP from nine subjects treated with gentamicin alone. These nine subjects were matched with the verapamil-treated subjects on the basis of gentamicin area-under-the-curve (AUC). After matching AUC, gentamicin exposure was virtually identical between the two groups with an average gentamicin AUC of 26.61 +/- 1.49 and 27.51 +/- 1.25 mg.hr/L for the gentamicin/ve-rapamil and gentamicin only groups respectively. Verapamil retarded the rise in mean daily AAP excretion on days 1 to 6 of gentamicin therapy, with a significant difference with respect to AAP urinary excretion (P < .05) observed on day 2. There was no significant difference in total cumulative AAP excretion or in the time to return to baseline AAP excretion. Therefore, verapamil was effective in reducing AAP excretion associated with gentamicin therapy.

Adolescent↗

Antioxidant and lysosomotropic properties of acridine-propranolol: protection against oxidative endothelial cell injury.

The antioxidant and lysosomotropic properties of a fluorescent analogue of propranolol, 9-amino-acridine-propranolol (9-AAP) were compared to those of propranolol. Using isolated microsomal membranes exposed to a superoxide and hydroxyl radical generating system, 9-AAP was found to be at least 10-fold more potent than propranolol (and about 50% as potent as vitamin E) in inhibiting lipid peroxidation. In cultured endothelial cells, 9-AAP afforded moderate protective effect against acute loss of glutathione but potent cytoprotective activity against free radical-mediated loss of viability/survival. Intracellular localization of 9-AAP was examined by fluorescent microscopy and compared with two known fluorescent lysosomal markers: acridine orange and Lysosensor. All three agents appeared to localize to similar peri-nuclear vesicles, presumably lysosomes or pre-lysosomes. Lysosensor fluorescence was not observable in the presence of 9-AAP, foreclosing the possibility of a direct dual labeling experiment. We employed the pH sensitivity of acridine orange to determine if it labels the same vesicles as 9-AAP. When the endothelial cells were preloaded with acridine orange, washed and resuspended in buffer containing 9-AAP, the dark orange-labeled vesicles observed with acridine orange alone became increasingly lighter with time. Since the fluorescence of acridine orange is altered by pH change, this spectral shift in fluorescence emission is consistent with the indication that added propranolol (or the analog) leads to lysosomal alkalization. In conclusion, 9-AAP is both a strong antioxidant and a lysosomotropic agent that is remarkably insensitive to photobleaching. These properties may contribute to the enhanced endothelial cytoprotective effects against free radical-induced injury.

Animals↗