GHB614 × T304-40 × GHB119 × COT102 Cotton: Protein Expression Analyses of Field-Grown Samples.

Food and feed safety assessment is not enhanced by performing protein expression analysis on stacked trait products. The expression levels of six proteins in cotton matrices from four single cotton events and three conventionally stacked trait cotton products are reported. Three proteins were for insect control; two proteins confer herbicide tolerance; and one protein was a transformation-selectable marker. The cotton matrices were produced at three U.S., five Brazil, and two Argentina field trials. Similar protein expression was observed for all six proteins in the stacked trait products and the single events. However, when two copies of the bar gene were present in the stacked trait products, the expression level of phosphinothricin acetyl transferase herbicide tolerance was additive. Conventional breeding of genetically engineered traits does not alter the level or pattern of expression of the newly introduced proteins, except when multiple copies of the same transgene are present.

Validity of an ELISA for N-acetyltransferase-2 (NAT2) phenotyping.

A competitive antigen ELISA was previously developed for NAT2 phenotyping, using caffeine as the probe drug. The ELISA phenotypes by measuring the ratio of 5-acetamido-6-amino-3-methyluracil (AAMU) and 1-methylxanthine (1X) after transformation of 5-acetamido-6-formylamino-3-methyluracil (AFMU) to AAMU, in contrast to capillary electrophoresis high-pressure liquid chromatography (HPLC) which phenotype by measuring the AFMU/1X ratio. The ELISA phenotyping was previously determined in 30 samples and correlated well with phenotypes determined by capillary electrophoresis (29/30). The correlation was extended with the standard HPLC methodology by expanding the data set by 146 in order to test the validity of the ELISA methodology. The correlation with HPLC in this larger sample size was 96%; whereas the correlation between the two methods for determination of 1X was high (r(2)=0.90), that for determination of AAMU by ELISA and AFMU by HPLC was low (r(2)=0.53). The poor correlation between the two methodologies could not be attributed to the age of urine samples, nor to a significant decomposition of AFMU in the body prior to collection of the urine sample. The addition of a simple caffeine metabolite extraction method, originally developed for HPLC analysis of metabolites, to the ELISA phenotyping protocol produced a methodology with absolute correlation to the standard HPLC method.

Cancer therapy and polymorphisms of cytochromes P450.

Cytochrome P450 (CYP) enzymes are important in the metabolism of some endogenous compounds, environmental and dietary xenobiotics and many drugs. Many of these enzymes have genetic polymorphisms that produce significant changes in metabolic activity, however the function of other polymorphisms is unknown. Genetic polymorphisms have important influences on variability in human pharmacokinetics, including intra-individual differences in drug toxicity, drug interactions and response to chemotherapy. Other factors that influence drug metabolism include differences in enzyme expression due to differences in age, gender, smoking status, exposure to dietary or environmental xenobiotics or co-administration of other drugs. In addition, some xenobiotics and drugs can directly inhibit or induce the activity of CYPs. All of these factors can produce differences in metabolic capacities among individuals which can produce toxicity in some patients and sub-effective dosing in others. Maximum clinical benefit will require a more complete understanding of the influence of these polymorphisms on allele function and their interaction with inducers and inhibitors of enzyme expression or activity. This effort will permit the pharmacogenetic screening of patients before the administration of drugs and result in the identification of individuals who are prone to adverse reactions or poor response, resulting in more effective individualized therapy.

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans.

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Agrin gene expression in mouse somatosensory cortical neurons during development in vivo and in cell culture.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

Differential expression of K4-AP currents and Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes.

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of cortical neurons. Here we describe the development of cortical neurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons.

The number of primary auditory afferents in the rat.

Published estimates of the number of primary auditory afferents in the rat differ by as much as 30%. We undertook to determine if the widely varying estimates were related to methodological differences, especially the difference between counting cells in Rosenthal's canal and fibers in the cochlear nerve. Type I ganglion cells and myelinated cochlear nerve fibers in the same ears were counted in Long-Evans and Sprague-Dawley strains. Type II spiral ganglion cells were also counted. In each strain the numbers of myelinated fibers and type I ganglion cells were essentially the same. Means for the Long-Evans were 18,036 fibers and 17,749 cells. Means for Sprague-Dawleys were higher: 19,444 fibers and 19,229 cells. The mean number of type II ganglion cells was also greater in Sprague-Dawley than in Long-Evans rats: 1,388 and 1,170, respectively. Cell and fiber counts from the two ears of the same animal differed on average by only 1%. The number of auditory afferents did not change with age over the range (2-10 months) studied here. Several methodological differences have probably contributed to the varying estimates of type I primary auditory afferents, but the discrepancies are not inherent in counts of fibers and spiral ganglion cells.

Rapid metabolic phenotypes for acetyltransferase and cytochrome P4501A2 and putative exposure to food-borne heterocyclic amines increase the risk for colorectal cancer or polyps.

The metabolic activation of food-borne heterocyclic amines to colon carcinogens in humans is hypothesized to occur via N-oxidation followed by O-acetylation to form the N-acetoxy arylamine that binds to DNA to give carcinogen-DNA adducts. These steps are catalyzed by hepatic cytochrome P4501A2 (CYP1A2) and acetyltransferase-2 (NAT-2), respectively, which are known to be polymorphic in humans. On the basis of this proposed metabolic activation pathway, patients at greatest risk to develop colorectal cancer or nonfamilial polyps should be those who possess both the rapid NAT-2 and rapid CYP1A2 phenotypes and are exposed to high dietary levels of carcinogenic heterocyclic amines. Using a method that involves caffeine administration and high pressure liquid chromatographic analysis of urinary metabolites, we have determined the CYP1A2 and NAT-2 phenotypes of 205 controls and 75 cancer/polyp cases. Exposure information was obtained using a dietary and health habits questionnaire. Both the rapid CYP1A2 and rapid NAT2 phenotypes were each slightly more prevalent in cases versus controls (57% and 52% versus 41% and 45%, respectively). However, the combined rapid CYP1A2-rapid NAT-2 phenotype was found in 35% of cases and only 16% of the controls, giving an odds ratio of 2.79 (P = 0.002). Univariate analysis of the questionnaire indicated that age, rapid-rapid phenotype, and consumption of well done red meat were associated with increased risk of colorectal neoplasia. Furthermore, a logistic regression model that included age (as a continuous variable), consumption of well done red meat, and rapid-rapid phenotype as independent covariates gave odds ratios of 1.08, 2.08, and 2.91, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The boutonniere deformity.

Understanding the pathophysiology of the boutonniere deformity requires a complete understanding of the anatomy of the dorsal tendon apparatus. This unique tendon mechanism often becomes unbalanced, requiring correction of its components. Splinting is the cornerstone of treatment for the boutonniere deformity. In the acute stage, splinting ensures that the continuity of the central tendon to its insertion into the middle phalanx is maintained, and in the chronic stage, its function is to correct the flexion contracture of the PIP joint and stretch the retinacular ligaments. Splinting is also important postoperatively because it permits healing of the central tendon and lateral bands in their correct anatomic positions. Without proper splinting, the patient with the boutonniere deformity could not be successfully treated. Frequently, surgery is necessary, and the choice of procedure depends on the stage of the condition and the extent of the defect in the extensor tendon mechanism. The procedure also depends on the success of the splinting program and stretching of the tight retinacular structures. If passive joint mobility can be restored and if tendon imbalance and retinacular tightness persist, rebalancing is necessary. This rebalancing can be accomplished by a tenotomy of the terminal extensor tendon, a lysis or release of the retinacular structures, or release of the insertion of the extensor tendon at the base of the proximal phalanx. Reconstituting the defect in the central tendon over the PIP joint is accomplished by using a variety of procedures, including mobilization and advancement of the more proximal portion of the central tendon, shifting the lateral bands, or a tendon graft.

Determination of CYP1A2 and NAT2 phenotypes in human populations by analysis of caffeine urinary metabolites.

The wide variations in urinary bladder and colo-rectal cancer incidence in humans have been attributed in part to metabolic factors associated with exposure to carcinogenic aromatic and heterocyclic amines. Cytochrome P-4501A2 (CYP1A2), which catalyses N-oxidation, and acetyltransferase (NAT2) which catalyses N- and O-acetylation, both appear to be polymorphically distributed in human populations; and slow and rapid NAT2 phenotypes have been implicated as risk factors for these cancers. Caffeine has also been shown to undergo 3-demethylation by CYP1A2, and it is further acetylated to 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by the polymorphic NAT2. In this report, we describe a metabolic phenotyping procedure that can be used to determine concomitantly the hepatic CYP1A2 and NAT2 phenotypes. For the NAT2 phenotype, we confirm the valid use of the urinary molar ratio of AFMU/1-methylxanthine, even in alkaline urines. For the CYP1A2 phenotype, the urinary molar ratio of [1,7-dimethylxanthine + 1,7-dimethyluric acid]/caffeine, taken at 4-5 h after caffeine ingestion, was identified from pharmacokinetic analyses of 12 subjects as being better correlated (r = 0.73; p = 0.007) with the rate constant for caffeine 3-demethylation than other previously suggested ratios. This procedure was then used to determine the CYP1A2 phenotype in subjects from Arkansas (n = 101), Italy (n = 95), and China (n = 78). Statistical and probit analyses of nonsmokers indicated that the CYP1A2 activity was not normally distributed and appeared trimodal. This trimodality allowed arbitrary designation of slow, intermediate, and rapid phenotypes, which ranged from 12-13% slow, 51-67% intermediate, and 20-37% rapid, in the different populations. A reproducibility study of 13 subjects over a 5 day or 5 week period showed that, with one exception, intraindividual variability did not alter this CYP1A2 phenotypic classification. Induction of CYP1A2 by cigarette smoking was also confirmed by the increased caffeine metabolite ratios observed in the Arkansas and Italian smokers (blonde tobacco). However, Italian smokers of black tobacco and Chinese smokers did not appear to be induced. Furthermore, probit analyses of Arkansas and Italian blonde tobacco smokers could not discriminate between phenotypes, apparently as a consequence of enzyme induction.

Treatment of skin loss in the hand.

In treating skin loss of the hand, the surgeon must be aware of the placement of skin and scars, the unique quality of palmar skin, the tendency of skin grafts to hyperpigment, the need for pain-free and well-padded amputation stumps that are free of painful neuromata and poorly padded phalangeal remnants. Skin grafts, palmar skin loss, regional flaps, distant flaps and local flaps are discussed. The histology and function characteristics of normal skin of the hand are reviewed.

Concurrent use of quinidine and disopyramide: evaluation of serum concentrations and electrocardiographic effects.

The effects of concurrent disopyramide phosphate and quinidine sulfate therapy were studied in 16 normal healthy adults. No adverse effects serious enough to warrant discontinuation of therapy were observed. There was a small but significant increase in disopyramide serum concentration when concurrent quinidine therapy was given and a small decrease in quinidine serum concentration when disopyramide was added. No significant change in elimination half-life was seen for either drug. Both drugs produced prolongation of the corrected QT interval. This QT prolongation was greater for quinidine than for disopyramide.

A phalangeal fracture model--quantitative analysis of rigidity and failure.

Nine types of internal fixation techniques were tested in 4-point bending using a pig metacarpal model for phalangeal fractures. Levels of bending rigidity and bending moments at failure were determined, and the modes of failure are described. Plate and screw fixation afforded the greatest rigidity, and epiphyseal fractures occurred, leaving intact the test section. Flexible wire loop fixation failed by wire cutting into bone when a square knot was used. Twisted wire unraveled when placed in tension. Depending on the fracture type and the wire placement. Kirschner wires failed either by slipping in the bone, twisting in the bone cortex, or bending at the bone cortex interface. Rigidity varied widely depending on the way in which the wires were employed.

Pitfalls in management of fingertip injuries and hand lacerations.

Fingertip injuries and lacerations of the hand are often dismissed as inconsequential, although this attitude can lead to more serious problems. Appropriate and timely treatment of these injuries prevents loss of function, preserves digital sensibility, and lessens the need for reconstructive surgery.

Mechanical analysis of Kirschner wire fixation in a phalangeal model.

Transverse divisions of the mid-shaft of freshly frozen pig metacarpals were fixed with Kirschner wires of two sizes and using four different configurations. Compared to the usual cross-pin fixation using wires of 0.889 mm. four wires (0.712 mm) eccentrically placed and with their ends hooked provided a 300% improvement in bending rigidity and 170% in bending movement.