Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase.

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of alpha-lactalbumin was determined using the IDRing assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 +/- 0.97 vs. 2.24 +/- 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 +/- 0.46 vs. 0.90 +/- 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the alpha-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.

Apolipoprotein E receptors and amyloid expression are modulated in an apolipoprotein E-dependent fashion in response to hippocampal deafferentation in rodent.

The entorhinal cortex lesion paradigm is a widely accepted and efficient method to provoke reactive synaptogenesis and terminal remodeling in the adult CNS. This approach has been used successfully to contrast the profile of reactivity from various proteins associated with Alzheimer's disease pathophysiology in wild-type and apolipoprotein E (apoE)-deficient (APOE ko) mice. Results indicate that the production of the beta-amyloid 1-40 peptide (A beta 40) is increased in response to neuronal injury, with a timing that is different between wild-type and APOE ko animals. Moreover, we report that baseline levels of the A beta 40 peptide are significantly higher in the APOE ko mice. The expression of the apolipoprotein E receptor type 2 (apoER2) is also modulated by the deafferentation process in the hippocampus, but only in APOE ko mice. These results provide novel insights as to the molecular mechanisms responsible for the poor plastic response reported in apoE4-expressing and apoE deficient mice in response to hippocampal injury.

Nerve conduction and sensorineural function in dental hygienists using high frequency ultrasound handpieces.

BACKGROUND: Oscillatory vibration from industrial power tools poses a well-recognized risk of peripheral nerve injury. There have been reports of elevated vibrotactile perception thresholds (VPT) among dentists, dental technicians, and dental hygienists, using rotary devices and ultrasonics. Elevated VPTs are an indicator of small fiber nerve or mechanoreceptor injury, but the high frequencies associated with dental instruments are presumed by the ISO to exceed physiological response upper thresholds. This study examines nerve conduction and sensorineural deficits in dental hygienists. METHODS: A cross-sectional study of 94 experienced dental hygienists was conducted to assess peripheral nerve function and clinical signs and symptoms. Specialized testing included measurement of VPTs for three different categories of mechanoreceptors, sensory nerve conduction tests with fractionated digit and palmar segments, and measurement of calibrated pinch force with force sensitive resistors (FSRs) during a simulated procedure. RESULTS: Chronic hand paresthesias were described by 44.7% of experienced dental hygienists. Sensory nerve conduction velocity (SNCV) across the wrist-palm segment of the median nerve. VPTs were particularly elevated at the FAII mechanoreceptor among experienced dental hygienists. Compared to participants without carpal tunnel syndrome (CTS), as defined by study criteria, 14 experienced hygienists with diagnosed CTS had almost twice the average weekly use of vibratory instruments -8.3 hr versus 4.5 hr, and had SNCV deficits along the digit -47.11 m/sec (+8.70) versus 42.57 m/sec (+8.25), and across the wrist -44.04 m/sec (+7.15) versus 41.36 m/sec (+9.27). There was a distinct subset of dental hygienists (27%) with a combination of low calibrated pinch force in simulations, subjective loss of strength and elevated VPTs, especially in the FAII mechanoreceptor population -110.82 db (+8.57) versus 104.84 db (+6.80) in the rest of the cohort. This subset also had a higher prevalence of paresthesias (67% vs. 39%) and greater cumulative vibration exposure (OR = 1.206 [CI 1.005-1.448]), than other hygienists. CONCLUSIONS: The high levels of paresthesias observed among dental hygienists appear to be attributable to several pathophysiological mechanisms, including, sensory nerve demyelination at the carpal tunnel and intrinsic to the digits, and dysfunction of fingertip mechanoreceptors. A distinct sub-population appears to exhibit a high level of accumulated abnormality.

Apolipoprotein C-I expression in the brain in Alzheimer's disease.

The H2 allele of apolipoprotein (apo) C-I is associated with Alzheimer's disease (AD). However, this association is potentially confounded by the linkage disequilibrium of H2 with the epsilon2 and epsilon4 alleles of apoE and of H1 with the epsilon3 allele. To establish plausibility for a direct role for apoC-I in AD, we compared apoC-I and apoE protein and mRNA levels in postmortem specimens of frontal cortex and hippocampus from AD patients with levels in nondemented controls. In H2-allelic individuals (usually also epsilon4 carriers), apoC-I mRNA levels were strikingly lower with AD (by 65%, P < 0.05), but apoC-I protein levels in AD were significantly higher (by 34%, P < 0.05). The opposite direction of the apoC-I mRNA and apoC-I protein level changes in AD in the epsilon4/H2 genotype may reflect decreased clearance of CNS lipoproteins associated with apoE4. In H1/H1 (usually epsilon3/epsilon3) individuals, both apoC-I protein and mRNA were lower in AD. ApoC-I protein levels in hippocampus were nearly twice those in frontal cortex. Immunohistochemistry of hippocampus revealed colocalization of apoC-I protein with the astrocytic marker GFAP. In addition, cultured human astrocytes expressed the mRNA for apoC-I. This study confirms apoC-I expression in the CNS and identifies astrocytes as the source of apoC-I. In addition, it has revealed differences in apoC-I expression based on site, genotype, and disease status that may reflect a role for apoC-I in the pathogenesis of AD.

Production and characterization of anti-nisin Z monoclonal antibodies: suitability for distinguishing active from inactive forms through a competitive enzyme immunoassay.

As a pre-requisite to monoclonal antibody development, an efficient purification strategy was devised that yielded 72 mg of nisin Z from 14.5 1 of Lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 (L. diacetylactis UL719) culture in supplemented whey permeate. Specific monoclonal antibodies (mAbs) were produced in mice against the purified nisin Z using keyhole limpet hemocyanin as a carrier protein. These antibodies did not recognize nisin A, suggesting that the asparagine residue at position 27 is involved in antibody recognition to nisin Z. However, the high reactivity of mAbs against biologically inactive nisin Z degradation products, produced during storage of freeze-dried pure nisin Z at -70 degrees C, indicated that the dehydroalanine residue at position 5 (Dha5), required for biological activity, is not necessary in nisin Z recognition by the mAb. A competitive enzyme immunoassay (cEIA) using the specific anti-nisin Z mAb was developed and used for rapid and sensitive detection and quantification of nisin Z in fresh culture supernatant, milk and whey. Detection limits of 78 ng/ml in phosphate-buffered saline, 87 ng/ml in culture supernatant, 106 ng/ml in milk and 90.5 ng/ml in whey were obtained for this assay. The cEIA using specific mAbs can be used to quantify nisin Z in food products.

Similar efficacy following four weeks treatment of asthmatics with formoterol 12 micrograms b.d. delivered by two different dry powder inhalers: differences in inhaler handling.

This randomised, multicentre, parallel-group study compared the clinical efficacy and ease of handling of two dry powder inhalers delivering the long-acting beta 2-agonist formoterol. After run-in, 200 asthmatics on treatment with inhaled corticosteroids and still presenting with suboptimal asthma control were randomised to receive 12 micrograms formoterol twice daily via either the Aerolizer inhaler (Foradil Aerolizer) or the Turbuhaler inhaler (Oxis Turbuhaler) for four weeks. Study variables included the mean morning pre-medication peak expiratory flow (PEF) during the last seven days of treatment and the correct inhaler handling according to inhaler-specific checklists. The mean difference in the effect on morning pre-medication PEF was 13.86 l/min in favour of formoterol via the Aerolizer inhaler (90% confidence interval 2.50, 25.21) in the intent-to-treat population. Eighty-six per cent of the patients under treatment with formoterol via the Turbuhaler inhaler performed correctly all the essential inhalation manoeuvres, whereas 98% of those on the Aerolizer inhaler did so. These results strongly suggest similar clinical efficacy with twice daily treatment of formoterol 12 micrograms metered dose delivered either by the Aerolizer, or the Turbuhaler device. They also suggest that handling the Aerolizer is easier than that of the Turbuhaler.

Coordinate regulation of neuroendocrine convertase PC2 and peptide 7B2 in P19 neurons.

Convertases are proteases responsible for the bioactivation of many proteins and peptides having a potential role in ontogenesis. As a model to study regulation of convertases in embryo, we use the P19 embryonal carcinoma cell line, which can differentiate into various cell types. The expression of convertase PC2 and its specific binding peptide 7B2 are co-induced during neuronal differentiation of P19 cells. We investigated the possibility that expression of both proteins may be coregulated by T3 and dexamethasone, activators of nuclear receptors, isobutylmethylxanthine, and dibutyryl cAMP, activators of protein kinase A, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. Western blotting results show that expression of PC2 and 7B2 can be upregulated by modulators of the protein kinases, and upregulation needs not be strictly stoichiometric.

Simultaneous immunofluorescent detection of coentrapped cells in gel beads.

An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.

Controlling dental hygiene work-related musculoskeletal disorders: the ergonomic process.

Work-related musculoskeletal disorders (WMSDs) have been reported frequently by dental hygienists. The practice of dental hygiene exposes practitioners to WMSD risk factors of repetitive motion, pinch-grasp, force, vibration, and prolonged awkward positions. The objective of an ergonomic program is to fit the job to the worker, rather than the worker to the work. Principles of the ergonomic process can be applied in private dental practice to design a program that meets the needs of each practitioner. The ergonomic process includes data collection, assessment, the ergonomic plan itself, and evaluation of strategies. The worker (dental hygienist), work processes, and the work environment are integral parts in the ergonomic process. The purpose of this article is to provide basic information on the ergonomic process so that individuals can develop a program that meets their need to reduce WMSDs.

Apolipoprotein E and beta-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependent.

The epsilon4 allele of apolipoprotein E (apoE) is associated with increased risk for the development of Alzheimer's disease (AD), possibly due to interactions with the beta-amyloid (Abeta) protein. The mechanism by which these two proteins are linked to AD is still unclear. To further assess their potential relationship with the disease, we have determined levels of apoE and Abeta isoforms from three brain regions of neuropathologically confirmed AD and non-AD tissue. In two brain regions affected by AD neuropathology, the hippocampus and frontal cortex, apoE levels were found to be decreased while Abeta(1-40) levels were increased. Levels of apoE were unchanged in AD cerebellum. Furthermore, levels of apoE and Abeta(1-40) were found to be apoE genotype dependent, with lowest levels of apoE and highest levels of Abeta(1-40) occurring in epsilon4 allele carriers. These results suggest that reduction in apoE levels may give rise to increased deposition of amyloid peptides in AD brain.

Production and characterization of polyclonal antibodies against cholecalciferol (vitamin D3).

Vitamin D is one of the essential vitamins in the human diet for normal growth and function. In Canada and the USA, fortified milk and milk products are the essential source of vitamin D. The adult recommended nutrient intake of vitamin D is 200 to 400 I.U. (corresponding to 5 to 10 microg) per day. Additional amounts of vitamin D do not confer benefits and may even be toxic. However, a deficiency of this vitamin leads to inadequate absorption of calcium and phosphorus and faulty mineralization of bones and teeth. Actual methods for measuring vitamin D in milk are limited in terms of sensitivity, rapidity and simplicity. The objective of this manuscript was to develop a new molecular strategy for the production, purification and characterization of polyclonal antibodies to vitamin D. Specific antibodies were raised in rabbits against vitamin D using cationized bovine serum albumin (cBSA) as a carrier protein. Anti-vitamin D antibodies were recovered from rabbit sera by sequential affinity chromatographies through Protein A/G Agarose, cBSA Sepharose and cOVA-vitamin D Sepharose columns. Although the yields of anti-vitamin D were relatively low, recovered antibodies showed high specificity and affinity to vitamin D. The purified antibody was used to develop a solid-phase enzyme immunoassay in order to determine the exact concentration of vitamin D in phosphate buffer. Using this immunoassay, approximately 35 ng of vitamin D can be detected within 3 h. The signal obtained was proportional to the amount of vitamin D in the sample analyzed. The strategy developed in this paper appears to be very promising in terms of sensitivity, rapidity and simplicity. It offers a great potential for automation and use on a routine basis for the quantification of vitamin D in fortified milk and other milk products.

Brain lipoprotein metabolism and its relation to neurodegenerative disease.

Lipoproteins are macromolecular complexes composed of lipids and proteins. The role of these complexes is to provide cells of the organism with lipids to be used as a source of energy, building blocks for biomembrane synthesis, and lipophilic molecules (e.g., steroid hormones and vitamin E) for other physiological purposes, such as cell signaling and antioxidative mechanisms. Lipoproteins also promote the cellular efflux of cholesterol for its disposal into bile. Thus, lipoproteins play an important role in the maintenance of lipid homeostasis throughout the organism. Accordingly, lipoprotein particles have been found circulating in blood, lymph, and interstitial fluid. Despite the existence of the blood-brain barrier, lipoprotein particles have been shown to be also present in the cerebrospinal fluid (CSF). Although a portion of their protein components may filter through the barrier from the vascular compartment, experimental evidence indicates that these particles originate from the nervous tissue. The other protein components include apolipoproteins E, J, and D, and these have been shown to be synthesized by cells within the central nervous system (CNS). Furthermore, it was shown that lipoprotein particles can be isolated from the conditioned medium of astrocytic cultures. The differences in size, structure, and composition of in vitro assembled particles compared with those isolated from the CSF suggest that the particles are modified following their secretion in vivo. This is supported by observations that lipoprotein-modifying enzymes and transfer proteins are also present within CNS tissue and CSF. The fate of CSF lipoproteins is unclear but is probably related to the turnover and clearance of lipids from the CNS or, alternatively, the particles may be recaptured and recycled back into the CNS tissue. The presence of several cell surface receptors for apoE-containing lipoproteins on ependymal cells, as well as on neurons and glial cells, supports this notion and suggests that the isolated brain possesses its own system to maintain local lipid homeostasis. This is further exemplified by the salvage and recycling of lipids shown to occur following a lesion in order to allow surviving neurons to sprout and reestablish lost synapses. Not much is currently known about lipoprotein metabolism in neurodegenerative diseases, but lipid alterations have been repeatedly reported in Alzheimer brains in which neuronal loss and deafferentation are major features. Although the mechanism underlying the link between the epsilon4 allele of the apolipoprotein E gene and Alzheimer's disease is presently unclear, it may well be postulated that it is related to disturbances in brain lipoprotein metabolism.

Convertase PC2 and the neuroendocrine polypeptide 7B2 are co-induced and processed during neuronal differentiation of P19 embryonal carcinoma cells.

Convertases of the subtilisin/kexin family are responsible for the biological activation of a variety of pro-proteins, pro-hormones, and pro-trophic factors, and thus can modulate various aspects of embryonic development. We investigated the expression of each convertase by Northern hybridization during cell differentiation in vitro, using the mouse embryonal carcinoma cell line P19 as a model. The neuroendocrine convertase PC2 and 7B2, its specific binding protein, are co-induced during neuronal differentiation of P19 cells with retinoic acid, whereas the other convertases are not or follow different patterns of temporal expression. The mature forms of PC2 and 7B2 proteins are detected together by immunoblotting following induction of mRNA expression, indicating that these proteins are processed early during brain development. These results demonstrate that PC2 and 7B2 gene expression and protein processing are in a close temporal association during neuronal differentiation and point to the value of the P19 cell model to study the significance and the regulation of this relationship in mammalian brain development.

Ergonomic strategies for dental professionals.

This study examined the extent to which dental professionals have made changes within their practice environments to decrease the potential for developing a cumulative trauma disorder. A survey was disseminated to a sample of 95 dental professionals. The survey addressed the presence and location of pain, changes professionals made within their workplaces, and whether or not these changes were perceived as effective. Fifty two surveys were returned for a response rate of 55%. Results indicated that 96% noted pain during or after work; 88% had made changes in their work practices. Common strategies used to promote health were stretching, good posture, personal relaxation, and instrument maintenance. Use of ergonomic instruments, workstations, and new instrumentation strategies were seen as effective, but rated slightly lower than other categories.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis.

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14-) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14- mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14- mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wild-type SEF14+ strain during the first week following infection as compared to those infected with the SEF14- mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected.

Characterization of recombinant tetanus toxin derivatives suitable for vaccine development.

Recombinant derivatives of tetanus toxin (TeTx) were isolated and used to immunize mice. Recombinant TeTx light chain, a derivative of fragment C that had lost the ability to bind neurons, and a recombinant TeTx holotoxoid that could protect mice against TeTx challenge were identified.

Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.

Cloning, DNA nucleotide sequence and distribution of the gene encoding the SEF14 fimbrial antigen of Salmonella enteritidis.

Monoclonal antibody 69/25, specific for the Salmonella enteritidis fimbrial antigen (SEF14), was used to screen a pUC-based S. enteritidis gene library and a positive clone was identified. Subcloning experiments demonstrated that a 584 bp DraI DNA fragment was the minimal chromosomal segment capable of directing SEF14 antigen expression. Western blotting of Escherichia coli recombinants identified a gene product of M(r) 16000 as a precursor to the M(r) 14300 mature fimbrial subunit protein. The DNA nucleotide sequence of the DraI fragment was determined and was shown to contain a single open reading frame with two potential f-Met start codons and a hydrophobic signal sequence. Downstream of a putative peptidase cleavage site, the deduced amino acid sequence showed considerable homology with the N-terminal amino acid sequence of what was originally described as the type 1 fimbrial subunit of Salmonella enteritidis and later redefined as SEF14. The gene encoding SEF14, designated as sefA, was shown to be limited in distribution to Salmonella blegdam, S. dublin, S. enteritidis, S. gallinarum, S. moscow, S. pullorum, S. rostock, S. seremban and S. typhi, all belonging to Salmonella group D. However, expression of the SEF14 antigen was limited to S. dublin, S. enteritidis, S. moscow and S. blegdam. The nucleotide sequence of the sefA gene shared no homology with the Salmonella fimA gene encoding type 1 fimbriae, and these genes showed distinct patterns of distribution within salmonellae.

Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

An evaluation of selected screening tests for bovine paratuberculosis.

The objective of this study was to evaluate the performance of the lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA), carbohydrate antigen complement fixation (CH-CFT), and protein D antigen agar gel immunodiffusion (D-AGID) tests for bovine paratuberculosis, relative to histopathology, and to culture and isolation of Mycobacterium paratuberculosis from tissues and feces. Samples for test evaluation were collected from four sources including blood and tissues from 400 cull cows at three abattoirs in Ontario, blood and feces from a paratuberculosis survey of cattle from 120 dairy farms in Ontario, a serum bank containing samples from cattle from Ontario and Québec, and a bank of sera from cattle from Pennsylvania and the northeastern United States. The data were analyzed using receiver operator characteristic curves, estimates of relative sensitivity and specificity, and kappa statistics of agreement between tests. The LAM-ELISA performed significantly better than both the CH-CFT and the D-AGID tests. The LAM-ELISA was better at predicting fecal shedding status than tissue infection. However, the LAM-ELISA also had limitations. When interpreted as positive or negative (+/-), at a critical optical density of 0.675, its sensitivity and specificity relative to bacteriology were 49% and 87% respectively. Although the serological tests examined in this study provided some information, they did not predict well the infection status of individual animals.