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Biomedical subjects

E J Kelly

Publications and source records attributed to E J Kelly.

At least 19 recordsLinked to original sources
PubMed2006

End-to-side nerve coaptation: a qualitative and quantitative assessment in the primate.

There are several reasons why end-to-side nerve coaptation has not been widely adopted clinically. Among these are the putative damage inflicted on the donor nerve and the variable quality of the regeneration in the recipient nerve. So far experiments on end-to-side nerve repair have been short term and mostly carried out on rats. This long-term study of end-to-side nerve repair of ulnar to median and median to ulnar nerve was performed using adult nonhuman primates. Eleven nerve repairs were studied at different time points. Eighteen, 22, 33 and 57 months after surgery a qualitative and quantitative analysis of the donor nerve and regenerating nerve revealed variable levels of percentage axonal regeneration compared with matched controls (1.4%-136%). Morphological evidence of donor nerve damage was identified distal to the coaptation site in four of the 11 cases, and in these cases the best axonal regeneration in the corresponding recipient nerves was observed. This donor nerve damage could neither be demonstrated in terms of a decrease in axon counts distal to the coaptation nor as donor target organ denervation. Recipient target organ regeneration like the axonal regeneration varied, with evidence of motor regeneration in eight out of 11 cases and sensory regeneration, as measured by percentage innervation density compared with matched controls, varied from 12.5% to 49%. Results from the present study demonstrate that the end-to-side coaptation technique in the nonhuman primate does not give predictable results. In general the motor recovery appeared better than the sensory and in those cases where donor nerve damage was observed there was better motor and sensory regeneration overall than in the remaining cases.

Animals↗
PubMed2005

Nerve fibre and sensory end organ density in the epidermis and papillary dermis of the human hand.

Quantification of sensory recovery after peripheral nerve surgery is difficult and no accurate techniques are available at present. Quantification of reinnervated skin has been used experimentally, and in some clinical studies, but the lack of knowledge about the normal sensory distribution has been a problem. The purpose of this study was, therefore, to map the density of sensory end organs, nerve fibres and free nerve endings in the glabrous skin of the human hand. Skin biopsies were taken from patients undergoing acute and elective hand surgery. Nerve fibres were stained in the epidermis and papillary dermis and quantified in five sites on the palm of the hand, using protein gene product 9.5 immunoreactivity-a panneuronal marker. The finger tip skin was found to have more than twice the nerve fibre density in the papillary dermis than the skin of the palm, and the number of Meissner corpuscles in the finger tip was also higher than in the palm. We found a reduction in innervation density with increasing age in the dermis, however, that was not the case for the epidermis. The innervation of the epidermis showed high interindividual variability and unlike the papillary dermis did not display any pattern of distribution in the hand.

Adult↗
PubMed2001

Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli.

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus. A single purification step employing metal (Ni(2+))-affinity column chromatography provided essentially homogeneous enzyme in yields of 69-72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 micromol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His(6)-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.

Acinetobacter↗
PubMed2001

Interindividual differences in response to chemoprotection against aflatoxin-induced hepatocarcinogenesis: implications for human biotransformation enzyme polymorphisms.

It is now evident that most, if not all, of the remarkable species differences in susceptibility to AFB hepatocarcinogenesis is due in large part, if not exclusively, to differences in biotransformation. Certainly the relative rate of oxidative formation of the proximate carcinogen, AFB-8,9-exo-epoxide, is an important determinant of species and interindividual differences in susceptibility to AFB. However, mice produce relatively large amounts of exo-AFBO, yet are highly resistant to AFB-hepatocarcinogenesis because they express a particular form of GST with remarkably high catalytic activity toward the exo-epoxide of AFB. Rats, which are highly susceptible to AFB hepatocarcinogenesis,can be made resistant through dietary induction of an orthologous form of GST that is normally expressed in only very small amounts. Based on these findings in laboratory animal models, there is great interest in identifying chemicals and/or specific dietary constituents that could offer protection against AFB-hepatocarcinogenesis to humans. Current experimental strategies have focused on the antiparasitic drug, oltipraz, which induces protection in rats and has also shown some promise in humans. The mechanism of protection in rats appears to be via induction of an alpha class GST with high catalytic activity toward AFBO (rGSTA5-5). vet human alpha class GST proteins that are constitutively expressed in the liver (hGSTA1 and hGSTA2) have little, if any activity toward AFBO. Rather, it appears that mu class GSTs may be responsible for the very low, but potentially significant, detoxification activity toward AFBO. Oltipraz and certain dietary constituents may induce mu class GSTs in human liver, and this could afford some protection against the genotoxic effects of AFBO. However, it also appears that oltipraz, and perhaps certain dietary constituents, act as competitive inhibitors of human CYP1A2. As CYP1A2 appears to mediate most of the activation of AFB to exo-AFBO in human liver at low dietary concentrations of AFB encountered in the human diet, much of the putative protective effects of oltipraz could be mediated via inhibition of CYP1A2 rather than induction of GSTs. There is now evidence that human microsomal epoxide hydrolase (mEH) could play a role in protecting human DNA from the genotoxic effects of AFB, although the importance of this detoxification pathway, relative to mu class GSTs, remains to be elucidated. Oltipraz is an effective inducer of mEH in rats (Lamb Franklin, 2000), and thus induction of this pathway in humans could also potentially contribute to the protective effects of this drug toward AFB genotoxicity. Because the dihydrodiol of AFB may contribute indirectly to the carcinogenic effects of AFB via protein adduction and subsequent hepatotoxicity, the recently characterized human aflatoxin aldehyde reductase (AFAR) may also offer some protection against AFB-induced carcinogenicity in humans. Current and future dietary and/or chemointervention strategies aimed at reducing the carcinogenic effects of AFB in humans should consider all of the possible mechanistic approaches for modifying AFB-induced genotoxicity.

Aflatoxin B1↗
PubMed2001

Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium.

BACKGROUND: The Toll protein in Drosophila regulates dorsal ventral patterning during embryogenesis, and participates in antibacterial and antifungal host defense. Mammalian homologues are termed Toll-like receptors and, to date, nine have been cloned (TLRI-9) in humans. They are characterized by extracellular leucine-rich repeats and a cytoplasmic domain similar to the interleukin 1 receptor. Both TLR2 and TLR4 recognize various bacterial cell wall components including lipopolysaccharide (LPS). This results in the activation of the NFkappaB pathway. Peripheral blood mononuclear cells (PBMCs) express both TLR2 and TLR4. The authors hypothesized that the expression of TLR 2 and TLR4 in human intestinal epithelial cells differs from PBMCs because of the abundance of LPS in the intestinal lumen. METHODS: Epithelial cells were isolated from Caco-2 cells, fetal gut explants, and small bowel resection specimens using Hanks/ethylenediamine tetraacetic acid solution. PBMCs were used as positive controls. Ribonucleic acid (RNA) was isolated using the TRIzol method. Standard reverse transcription-polymerase chain reaction examined TLR2 and TLR4 messenger RNA (mRNA) expression. NFkappaB expression was determined using a luciferase reporter assay. RESULTS: TLR2 mRNA was highly expressed in PBMCs and was present in all human intestinal epithelial cells. TLR4 mRNA was detected only in PBMCs. TLR4 is not present in epithelium from children with inflammatory bowel disease. In Caco-2 cells, significant NFkappaB activation in response to LPS occurred only in the presence of TLR4 introduced by complementary deoxyribonucleic acid transfection. CONCLUSION: Absence of TLR4 is associated with endotoxin hyporesponsiveness of intestinal epithelial cells. TLR4 is not directly involved in inflammation of the intestinal epithelium. Although TLR2 is normally present in the epithelial cell, it plays a limited role in inflammation. It may be activated during conditions in which bacterial cell wall concentrations within the intestine are pathologically high.

Adolescent↗
PubMed2001

Hepatic failure and hemochromatosis of Salers and Salers-cross cattle.

Hemochromatosis is rare in domestic mammals. Five clinical cases and one preclinical case of hemochromatosis were diagnosed in Salers and Salers-cross cattle. Clinical disease developed between 9 and 22 months of age. Animals were healthy until weaning but then lost weight, developed rough hair coats, and lost incisor teeth. In two animals, hemochromatosis was identified by liver biopsy, biochemical evidence of hepatic injury, and/or elevated transferrin saturation values. At necropsy, carcasses were thin, with firm dark brown livers and lymph nodes, soft bones, and brown-colored small bowel. The principal histologic changes were hepatocellular siderosis and periportal, bridging, and perivenular fibrosis. Siderocalcinosis involved collagen, elastin, reticulin, and basement membrane components in liver, lymph nodes, spleen, duodenum, and kidney. Hepatic iron concentrations in clinically affected cattle were 1,500-10,500 microg/g wet weight (reference range for cattle = <300 microg/ g). Ultrastructurally, the heaviest intrahepatic deposition was in hepatocytes, which contained large intracytoplasmic siderosomes. Iron deposition in bone was associated with osteopenia. Genetic analysis indicated a common ancestral bull in the pedigrees of five of six affected cattle; no pedigree was available for the remaining animal. Four dams of five affected animals were phenotypically normal and had histologically normal livers. Test mating of four cows to the ancestral bull resulted in a female calf that developed clinicopathologic and histologic evidence of preclinical hemochromatosis by 40 days of age. It was not possible to establish the pattern of inheritance because of the small number of pedigrees from affected cattle.

Animals↗
PubMed2001

Attitudes of parents of nondisabled students regarding inclusion of disabled students in Nevada's public schools.

Opinions of 370 parents of students without disabilities about inclusive programming were compared with those obtained from a previous study of 65 parents of students with disabilities. Both groups of parents positively rated opinion statements favoring inclusion; however, parents of students with disabilities consistently rated inclusion-focused statements more positively than parents of nondisabled students.

Adult↗
PubMed2000

ASHP national survey of pharmacy practice in acute care settings: monitoring, patient education, and wellness--2000.

Results of the 2000 ASHP national survey of pharmacy practice in acute care settings that pertain to patient medication monitoring, education, and wellness are presented. Pharmacy directors at 1063 general and children's medical-surgical hospitals in the United States were surveyed by mail. The response rate was 50.2%. Although the respondents indicated that most pharmacists spent less than 20% of their time on medication-monitoring activities, the amount of time devoted to such activities was increasing. Pharmacists were selective about which patients they chose to monitor for medication-related problems. Patients were frequently chosen on the basis of service or medication. Pharmacists used a number of mechanisms to monitor patients for adverse drug events (ADEs). Although internal ADE reporting had generally increased within the preceding three years, 81% of the Institutions had implemented strategies to improve reporting. When ADEs were reported externally (59% of the respondents), FDA was most commonly alerted. About 92% of the respondents indicated that nursing had primary responsibility for counseling patients about medications. Pharmacists were infrequently involved in medication education during the hospital stay; however, 48% of the institutions used some method to identify patients needing counseling by pharmacists. Slightly more than half of the respondents reported having wellness programs. Pharmacists were most commonly involved in disease-based wellness programs. Pharmacists in acute care settings appear to be well positioned to improve the patient-monitoring, education, and wellness components of the medication-use process.

Adverse Drug Reaction Reporting Systems↗
PubMed2000

Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver.

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.

Aflatoxin B1↗
PubMed1999

Microneurovascular transfer of contralateral latissimus dorsi in Poland's syndrome.

A 2-year-old girl with Poland's syndrome presented with syndactyly of the first, second and third webs of the left hand and ipsilateral absence of the sternocostal portion of pectoralis major. Latissimus dorsi on the same side was also absent. A microvascular free contralateral latissimus dorsi muscle transfer was undertaken to reconstruct the chest wall and anterior axillary fold. The transfer was innervated by intercostal nerve transfer and produced a neuromuscular unit of good aesthetic appearance and function.

Child, Preschool↗
PubMed1999

Nutrient regulation of intestinal gene expression.

This review examines recent advances in the dietary modulation of gene expression in the gastrointestinal tract. We have chosen to concentrate on individual genes and examine what is known about their regulation, attempting to link different studies together.

Animal Nutritional Physiological Phenomena↗
PubMed1998

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation.

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse-chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28 degreesC resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Adenosine Triphosphatases↗
PubMed1998

The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues.

Secretory mucins play an important role in the cytoprotection of epithelial surfaces and are used as tumour markers in a variety of cancers. The MUC6 secretory mucin was originally isolated from a gastric cDNA library. The aim was to determine the specific type and location of MUC6 mucin gene expression in a wide range of human adult and fetal epithelial tissues. In situ hybridization, RNA analysis, and immunohistochemistry were used to quantify and localize mucin gene expression. The data obtained show that MUC6 is highly expressed in gastric mucosa, duodenal Brunner's glands, gall bladder, seminal vesicle, pancreatic centroacinar cells and ducts, and periductal glands of the common bile duct; focal expression is seen in basal endometrial and endocervical glands. MUC6 epitopes were also highly expressed in 7/10 pancreatic cancers and 7/10 cholangiocarcinomas and focally expressed in 4/10 endocervical adenocarcinomas. Expression of MUC6 occurs early in fetal development and was observed in Brunner's glands and pancreatic ducts at 18-19 weeks and in gastric glands at 20 weeks' gestation. The tissue distribution of the MUC6 secretory mucin indicates that it may function to protect epithelial tissues from a wide range of substances. Expression of MUC6 is frequently preserved in pancreatic and bile duct adenocarcinomas, but it is only sparsely expressed in endocervical carcinomas.

Biomarkers, Tumor↗
PubMed1998

Ectopic expression of metallothionein-III causes pancreatic acinar cell necrosis in transgenic mice.

Mice express four distinct metallothioneins (MTs) that have similar metal-binding properties. MT-I and MT-II are expressed coordinately in most organs, whereas MT-III is expressed predominantly in a subset of neurons and MT-IV is expressed in certain stratified epithelia. The restricted expression of MT-III suggests that it may severe a specialized function. To test this hypothesis, transgenic mice were generated that express MT-III in the wider expression domain of MT-I. Similar transgenic lines expressing extra MT-I under the same regulation were generated as controls for the effect of over-expression of MT. Transgenic mice that express MT-III ectopically frequently die at 2-3 months of age. The pancreata of moribund mice were abnormally small and histological examination, at various ages, revealed a progressive degeneration of the acinar cells. At early stages multifocal acinar cell eosinophilia and swollen nuclei were seen and this pathology progressed to multifocal acinar cell necrosis and fibrosis. The terminal stages were characterized by a loss of the acinar compartment, leaving the islets embedded in a fibrotic remnant. Other organs of these mice were grossly and histologically normal. All organs examined from mice expressing excess MT-I were unremarkable even though expression of either MT-I or MT-III transgenes resulted in similar accumulations of zinc and copper in the pancreata. This study indicates that pancreatic acinar cells are unusually sensitive to chronic expression of MT-III. The mechanism by which MT-III disrupts pancreatic function is unclear, but the results provide further evidence that MT isoforms exhibit distinct properties and probably serve distinct biological functions.

Animals↗
PubMed1998

Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad. Sci. USA 93, 1671-1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted ove the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, i.p.) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (> 90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 doses not play a significant role in APAP hepatotoxicity in these mice.

Acetaminophen↗
PubMed1998

An anomalous muscle in the hand.

An anomalous muscle crossing the wrist joint is described. The morphology and clinical relevance of this and other unnamed muscles occurring at the wrist are discussed.

Adult↗