Swine in biomedical research: creating the building blocks of animal models

The opportunities for utilizing swine biomedical models are immense, particularly in models that address lifestyle issues (nutrition, stress, alcohol, drugs of abuse, etc.). However, in order to fully capitalize upon the promise, there needs to be a more general recognition of these cofactors, such as nutrition, as key modulators of phenotype via genomic, epigenetic, and postgenomic mechanisms. Furthermore, increased interactions between nutrition scientists and clinical and fundamental researchers in other disciplines, including developmental biology, immunology, neuroscience, oncology, and cardiovascular and gastrointestinal physiology, are required. Closing discussions focused on the need for future conferences at more frequent intervals to support interactions between the various disciplines. This was especially critical because of the global distribution of investigators.     

MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth

The three known inhibitors of axonal regeneration present in myelin--MAG, Nogo, and OMgp--all interact with the same receptor complex to effect inhibition via protein kinase C (PKC)-dependent activation of the small GTPase Rho. The transducing component of this receptor complex is the p75 neurotrophin receptor. Here we show that MAG binding to cerebellar neurons induces alpha- and then gamma-secretase proteolytic cleavage of p75, in a protein kinase C-dependent manner, and that this cleavage is necessary for both activation of Rho and inhibition of neurite outgrowth.     

Relationships between quantitative and reproductive fitness traits in animals

The relationships between quantitative and reproductive fitness traits in animals are of general biological importance for the development of population genetic models and our understanding of evolution, and of great direct economical importance in the breeding of farm animals. Two well investigated quantitative traits--body weight (BW) and litter size (LS)--were chosen as the focus of our review. The genetic relationships between them are reviewed in fishes and several mammalian species. We have focused especially on mice where data are most abundant. In mice, many individual genes influencing these traits have been identified, and numerous quantitative trait loci (QTL) located. The extensive data on both unselected and selected mouse populations, with some characterized for more than 100 generations, allow a thorough investigation of the dynamics of this relationship during the process of selection. Although there is a substantial positive genetic correlation between both traits in unselected populations, caused mainly by the high correlation between BW and ovulation rate, that correlation apparently declines during selection and therefore does not restrict a relatively independent development of both traits. The importance of these findings for overall reproductive fitness and its change during selection is discussed.     

The endothelial glycocalyx: a potential barrier between health and vascular disease

PURPOSE OF REVIEW: Although cardiovascular prevention has improved substantially, we still face the challenge of finding new targets to reduce the sequelae of atherosclerosis further. In this regard, optimizing the vasculoprotective effects of the vessel wall itself warrants intensive research. In particular, the endothelial glycocalyx, consisting of proteoglycans, glycoproteins and adsorbed plasma proteins, may play an essential role in protecting the vessel wall from atherosclerosis. RECENT DEVELOPMENTS: In this review, we will discuss the different vasculoprotective effects exerted by the endothelial glycocalyx, the factors that damage it, and the first preliminary data on the glycocalyx dimension in humans. Whereas most glycocalyx research has traditionally focused on the microvasculature, more recent data have underscored the importance of the glycocalyx in protecting the macrovasculature against pro-atherogenic insults. It has been shown that glycocalyx loss is accompanied by a wide array of unfavourable changes in both small and larger vessels. Pro-atherogenic stimuli increase the shedding of glycocalyx constituents into the circulation, contributing to the progressive loss of the vasculoprotective properties of the vessel wall. Novel techniques have facilitated reproducible measurements of systemic glycocalyx volume in humans. Consistent with experimental data, the volume of the human glycocalyx is also severely perturbed by exposure to atherogenic risk factors. SUMMARY: Cumulating evidence suggests that an intact glycocalyx protects the vessel wall, whereas disruption of the glycocalyx upon atherogenic stimuli increases vascular vulnerability for atherogenesis.     

Case report: Cerebral cholesterol granuloma in homozygous familial hypercholesterolemia

Familial hypercholesterolemia (FH) is characterized by the accumulation of excess cholesterol in tissues including the artery wall and tendons. We describe a patient with homozygous FH who presented with asymptomatic cholesterol granuloma of the brain. The patient''s plasma low-density lipoprotein cholesterol level was remarkably responsive to combination hypolipidemic therapy with statin plus ezetimibe. This case illustrates another potential complication of whole-body cholesterol excess and underscores the differences in phenotype and in response to therapy among patients with FH.A 45-year-old Chinese-Canadian woman presented with chemosis of several years'' duration. As a teenager the patient had noticed corneal arcus and thickening of her Achilles tendons. At age 20 she was found to have a fasting total cholesterol level of 18.0 (normal < 5.2) mmol/L and a low-density lipoprotein (LDL) cholesterol level of 15.4 (normal < 3.4) mmol/L. On the basis of these findings and a family history of hypercholesterolemia in both parents and premature coronary artery disease in her father, homozygous familial hypercholesterolemia (FH) was diagnosed. The initial treatment was with bile acid-binding resins, followed by the addition of hydroxymethylglutaryl coenzyme A reductase inhibitors, or statins, when she was 29. She has been intolerant of fibrates and niacin, both of which caused rashes. At age 30, angina pectoris necessitated coronary artery bypass grafting, and she has since remained free of cardiovascular symptoms. At age 40, carotid ultrasound imaging, conducted after carotid bruits were detected, revealed more than 95% and 50%–79% stenoses of the left and right carotid arteries respectively. She began taking 325 mg of ASA daily and has remained free of neurologic symptoms. Her total cholesterol level and LDL cholesterol level fell to 10.1 and 8.2 mmol/L respectively after she began taking 80 mg of atorvastatin and 4 g of colestipol daily, and her Achilles tendon xanthomas also shrank over time. Her lipoprotein (a) level was elevated at 0.44 (normal < 0.3) g/L. Her sitosterol level was 2.4 (normal ≤ 5.0) μg/mL, and her homocysteine level was 8.0 (normal < 12.1) μmol/L); her ApoE genotype (ε3/ε3) was also normal. The patient''s high-density lipoprotein cholesterol level was 0.8 (normal > 0.9) mmol/L on no lipid therapy and has ranged between 0.6 and 1.1 mmol/L with atorvastatin and colestipol therapy; her triglyceride level was 2.7 (normal < 2.3) mmol/L at baseline and has ranged from 1.4 to 3.6 mmol/L with therapy.A genomic DNA sequence analysis showed that she was homozygous for proline-to-leucine substitution at residue 644 of the LDL receptor known as FH-Zambia¹ or FH-Gujerat,²which has not been reported in Canada.³ This mutation is associated with slowed maturation and attenuated cell-surface expression of LDL receptors⁴ but does not abolish receptor function. Partial LDL receptor function appears to explain the patient''s responsiveness to therapy, which was better than expected for most patients with homozygous FH. The addition of 10 mg daily of ezetimibe to the atorvastatin and colestipol therapy lowered the patient''s LDL cholesterol level to 6.9 mmol/L. When colestipol was discontinued, this level decreased further to 5.9 mmol/L, which suggests an enhanced response to combination statin–ezetimibe therapy in the absence of bile acid-binding resin. Fig. 1 shows the sites of action of medications taken by the patient. We are considering LDL apheresis to further decrease LDL levels.Open in a separate windowFig. 1: Sites of action of medications taken by the patient. The liver is central to cholesterol metabolism. Hepatic cholesterol can be synthesized from acetyl coenzyme A (CoA) in a multistep enzymatic process, whose rate-limiting enzyme (3-hydroxy-3-methylglutaryl CoA reductase) is inhibited by statins such as atorvastatin. Hepatic cholesterol is used in part to synthesize bile acids that are destined, with sterols, for secretion in bile. The luminal sterol pool in the upper portion of the small intestine comes from both dietary and biliary sterols. Intestinal luminal sterols are transported into enterocytes and repackaged into chylomicrons for secretion into lymph and, ultimately, plasma. Ezetimibe likely inhibits sterol absorption by interfering with the sterol transporter Niemann–Pick C1 Like 1 protein.⁵ Bile acids advance through the intestinal lumen and facilitate intestinal cholesterol absorption. Unbound bile acids are normally recycled through the terminal ileum into the enterohepatic circulation. Bile acid-binding resins, such as colestipol, prevent this recycling and force more hepatic cholesterol into bile acid synthesis. To compensate for the depletion of hepatic cholesterol stores induced by all of these drugs, the liver increases expression of cell-surface low-density lipoprotein (LDL) receptors to extract more cholesterol from the plasma by receptor-mediated endocytosis. Although both copies of the LDL-receptor gene were defective in this patient — causing very high baseline plasma LDL cholesterol — each had some residual activity, and she was able to respond to treatment. See the animated figure at www.cmaj.ca/cgi/content/full/172/4/495/DC1. Photo: Lianne Friesen and Nicholas WoolridgeOphthalmologic examination revealed bilateral chemosis and conjunctival redness with no ocular hypertension or congested retinal vasculature. The patient was clinically and biochemically euthyroid, with no thyroid antibodies. Although the chemosis was felt to have resulted from chronic use of vasoconstricting eye whiteners, a head MRI scan was performed to rule out thyroid ophthalmopathy. It revealed an incidental large calcified mass involving the skull and meninges of the left temporal-occipital area (Fig. 2). Craniotomy for the resection of a presumed meningioma revealed a large, encapsulated, avascular mass emanating from the petrous bone that was filled with necrotic tan-to-yellowish paste-like material. Microscopy of the lesion''s capsule showed cholesterol clefts with granulomatous tissue including lipid-containing macrophages, multinucleated giant cells, chronic inflammation and fibrosis, with the contents showing shards of cholesterol and foci of calcification, consistent with a cholesterol granuloma. The patient had no perioperative complications or neurologic sequelae. A head CT scan performed 18 months after the operation showed no residual or recurrent mass, which is typical following resection of these tumours if the petrous bone is properly pneumatized.⁶ The chemosis persists, but we feel that, rather than being related to the tumour, it is a consequence of rebound vasodilatation secondary to chronic use of eye whiteners, even though the patient has stopped using them.Open in a separate windowFig. 2: MRI of a large intracranial cholesterol granuloma complicating homozygous familial hypercholesterolemia.Most cerebral cholesterol granulomata have not been associated with genetic hypercholesterolemia,^7,8 with only 1 previous case linked to homozygous FH.⁹ These lesions arise silently and are thought to occur as a foreign body reaction to cholesterol crystals deposited in or adjacent to the mastoid air cells following blockage of the air cell system, in which negative pressure and hypoxia lead to local tissue and blood cell necrosis.⁷ Cholesterol granulomata occur in about 1 per 2–3 million population each year.^8,10 They frequently cause neurosensory hearing loss or cranial nerve palsies or both,^7,8 but our patient had neither. She also had no history of known antecedents to cholesterol granulomata, including cholesteatoma, otitis media or cerebral bleeding.⁷ It seems surprising that cholesterol granulomata have not been more commonly reported in cases of homozygous FH, a condition with whole-body cholesterol excess.¹ The absence of neurologic symptoms despite the large intracranial mass was likely a result of the very slow growth of the granuloma. The constellation of an asymptomatic cerebral cholesterol granuloma, comparatively late-onset cardiovascular disease and relatively good response to hypolipidemic therapy underscores the phenotypic heterogeneity among patients with homozygous FH. Cerebral cholesterol granulomata should be added to the list of potential complications of FH.     

Aspects of potato leafhopper (Homoptera: Cicadellidae) biology on Solanum berthaultii and other potato genotypes

Survival of nymphs and adult males and females of potato leafhopper, Empoasca fabae (Harris), on six potato (Solanum spp.) genotypes with varied levels of resistance was evaluated and related to type, density, and droplet/head size of foliar glandular trichomes. Survival was greater on Solanum tuberosum cultivars ('Allegany' and 'Elba') compared with accessions of the wild S. berthaultii (PI 473331 and PI 473334). The barrier provided by glandular trichomes may affect survival of E.fabae by restricting nymph and adult feeding. PI 473331 was the most pubescent host, bearing type A and type B trichomes and the most unsuitable host for E. fabae. Allegany possesses leaflets with low trichome densities and small gland diameter and was the most suitable host for adult E. fabae survival. Females generally had higher survival rates than males. Nymphal and adult leafhoppers differed greatly in their survival rates, particularly on S. tuberosum x S. berthaultii hybrids (Q174-2 and NY123). Interestingly, nymphs had longer (190 d) mean lethal time (LT50) than adults (31 d) when held on Q174-2, and a lower LT50 (19 d) when held on NY123 compared with adults (104 d). Starvation, dehydration, or both were likely associated with mortality of nymphs confined on PI 473331 and PI 473334. Nymphs on these accessions died at a comparable rate to those confined on a starvation treatment and more rapidly than those supplied with water only, sucrose solution or other potato genotypes. Darkened trichome exudates were observed on insects held on PI 473331 and PI 473334, consistent with previous reports that glandular trichomes of S. berthaultii contain phenolic oxidation chemistry. This study aids in the identification of the probable mechanisms of resistance to E. fabae used by S. berthaulti and its hybrids with S. tuberosum.     

Amplifier spurious input current components in electrode-electrolyte interface impedance measurements

Background  

In Impedance Microbiology, the time during which the measuring equipment is connected to the bipolar cells is rather long, usually between 6 to 24 hrs for microorganisms with duplication times in the order of less than one hour and concentrations ranging from 10¹ to 10⁷ [CFU/ml]. Under these conditions, the electrode-electrolyte interface impedance may show a slow drift of about 2%/hr. By and large, growth curves superimposed on such drift do not stabilize, are less reproducible, and keep on distorting all over the measurement of the temporal reactive or resistive records due to interface changes, in turn originated in bacterial activity. This problem has been found when growth curves were obtained by means of impedance analyzers or with impedance bridges using different types of operational amplifiers.     

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.