Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Abnormal extracellular matrix protein transport associated with increased apoptosis of vascular smooth muscle cells in Marfan syndrome and bicuspid aortic valve thoracic aortic aneurysm

Background - Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)- 2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results - Aneurysm tissue was obtained from patients undergoing surgery ( MS: 4 M, 1 F, age 27 - 45 years; BAV: 3 M, 2 F, age 28 - 65 years). Normal aorta from subjects with nonaneurysm disease was also collected ( 4 M, 1 F, age 23 - 93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs. There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS ( 27 +/- 8%) and BAV ( 32 +/- 14%) compared with control ( 7 +/- 5%). Conclusions - In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis

Background The mechanisms responsible for disturbed iron homoeostasis in hereditary haemochromatosis are poorly understood. However, results of some studies indicate a link between hepcidin, a liver-derived peptide, and intestinal iron absorption, suggesting that this molecule could play a part in hepatic iron overload. To investigate this possible association, we studied the hepatic expression of the gene for hepcidin (HAMP) and a gene important in iron transport (IREG1) in patients with haemochromatosis, in normal controls, and in Hfe-knockout mice. Methods We extracted total RNA from the liver tissue of 27 patients with HFE-associated haemochromatosis, seven transplant donors (controls), and Hfe-knockout mice. HAMP and IREG1 mRNA concentrations were examined by ribonuclease protection assays and expressed relative to the housekeeping gene GAPD. Findings There was a significant decrease in HAMP expression in untreated patients compared with controls (5.4-fold, 95% CI 3.3-7.5; p

Cytokinin-induced abnormal shoot organogenesis is associated with elevated Knotted1-type homeobox gene expression in tobacco

The molecular mechanisms that regulate the transcription of key developmental genes involved in shoot organogenesis have yet to be fully elucidated. However, it is clear that plant growth regulators, such as cytokinin, play a critical role in the differentiation of adventitious shoots. In Nicotiana tabacum zz100 leaf discs, high frequency shoot formation could be induced with 5 muM of the cytokinin N-6-benzyladenine (BA). Increasing the exogenous BA concentration to greater than 20 muM resulted in stunted explants with abnormal shoot morphology and altered mineral composition. Explants with abnormal shoots did not appear to be hyperhydric. Abnormalities were, however, associated with an increase in the expression of a knotted1-type homeobox gene (TobH1) isolated from normal shoot-forming cultures. The results suggest that the development of cytokinin-induced abnormal shoot morphology possibly involves changes in TobH1 gene expression.

Alterations of left ventricular myocardial characteristics associated with obesity

Background-Obesity is associated with heart failure, but an effect of weight, independent of comorbidities, on cardiac structure and function is not well established. We sought whether body mass index (BMI) and insulin levels were associated with subclinical myocardial disturbances. Methods and Results-Transthoracic echocardiography, myocardial Doppler-derived systolic (sm) and early diastolic velocity ( em), strain and strain rate imaging and tissue characterization with cyclic variation (CVIB), and calibrated integrated backscatter (cIB) were obtained in 109 overweight or obese subjects and 33 referents (BMI35) and the referent patients (P

Tissue Doppler imaging for evaluation of myocardial function in patients with diabetes mellitus

Purpose of review Heart failure and diabetes mellitus are frequently associated, and diabetes appears to potentiate the clinical presentation of heart failure related to other causes. The purpose of this review is to examine recent advances in the application of tissue Doppler imaging for the assessment of diabetic heart disease. Recent findings Recent studies have documented that both myocardial systolic and diastolic abnormalities can be identified in apparently healthy patients with diabetes and no overt cardiac dysfunction. Interestingly, these are disturbances of longitudinal function, with compensatory increases of radial function-suggesting primary involvement of the subendocardium, which is a hallmark of myocardial ischemia. Despite this, there is limited evidence that diabetic microangiopathy is responsible-with reduced myocardial blood volume rather than reduced resting flow, and at least some evidence suggesting a normal increment of tissue velocity with stress. Finally, a few correlative studies have shown association of diabetic myocardial disease with poor glycemic control, while angiotensin converting enzyme inhibition may be protective. Summary Tissue Doppler imaging (and the related technique of strain rate imaging) appears to be extremely effective for the identification of subclinical LV dysfunction in diabetic patients It is hoped that the recognition of this condition will prompt specific therapy to prevent the development of overt LV dysfunction.

Pathology associated with endogenous development of haematozoa in birds from southeast Queensland

A study was undertaken on the pathology and associated schizont morphology of apicomplexan species of avian haematozoa. Some 32 birds from the families Artamidae, Meliphagidae, Oriolidae, Podargidae, Columbidae, Alcedinidae and Psittacidae were identified as having schizonts in various tissues. Based on blood stages observed, the probable relationship to tissue stages was considered. The majority of schizonts were referable to the genera Leucocytozoon and Haemoproteus . The comparative morphology of tissue stages previously described in the literature is discussed and the involvement of protozoa other than haematozoa considered. The naturally occurring infections in wild birds described in this study represent previously unreported data on the life-cycle stages involved. Some schizonts measured up to 640 mum. While pathological changes in some hosts were noticeable, in others no significant findings were observed. The role of endogenous stages in avian morbidity is discussed briefly.

Benefits of ACE inhibitors with tissue-active levels in the cardiac-compromised patient

Angiotensin converting enzyme inhibitors (ACEI) have been proven beneficial to the cardiac-compromised patient, but whether there is an advantage associated with using a tissue-active or systemically-active ACEI is debatable. An investigation into the clinical benefits of tissue ACEI for veterinary patients was undertaken by comparing enalapril with ramipril. Results obtained concluded that although there is much evidence to prove that tissue ACEIs are superior over systemic ACEIs at the cellular level, this does not correlate in the clinical sense. Both enalapril and ramipril provided similar clinical benefits to the cardiac-compromised patient.

The estrogen receptor 1 G594A polymorphism is associated with migraine susceptibility in two independent case/control groups

Migraine is a painful and debilitating disorder with a significant genetic component. Steroid hormones, in particular estrogen, have long been considered to play a role in migraine, as variations in hormone levels are associated with migraine onset in many sufferers of the disorder. Steroid hormones mediate their activity via hormone receptors, which have a wide tissue distribution. Estrogen receptors have been localized to the brain in regions considered to be involved in migraine pathogenesis. Hence it is possible that genetic variation in the estrogen receptor gene may play a role in migraine susceptibility. This study thus examined the estrogen receptor 1 (ESRalpha) gene for a potential role in migraine pathogenesis and susceptibility. A population-based cohort of 224 migraine sufferers and 224 matched controls were genotyped for the G594A polymorphism located in exon 8 of the ESR1 gene. Statistical analysis indicated a significant difference between migraineurs and non-migraineurs in both the allele frequencies (P=0.003) and genotype distributions (P=0.008) in this sample. An independent follow-up study was then undertaken using this marker in an additional population-based cohort of 260 migraine sufferers and 260 matched controls. This resulted in a significant association between the two groups with regard to allele frequencies (P=8x10(-6)) and genotype distributions (P=4x10(-5)). Our findings support the hypothesis that genetic variation in hormone receptors, in particular the ESR1 gene, may play a role in migraine.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

Low level microsatellite instability may be associated with reduced cancer specific survival in sporadic stage C colorectal carcinoma

Background: Colorectal cancers (CRCs) may be categorised according to the degree of microsatellite instability (MSI) exhibited, as MSI-high (MSI-H), MSI-low (MSI-L), or microsatellite stable (MSS). MSI-H status confers a survival advantage to patients with sporadic CRC. Aims: To determine if low levels of MSI are related to the clinicopathological features and prognosis of sporadic stage C CRC. Patients: A total of 255 patients who underwent resection for sporadic stage C CRC were studied. No patient received chemotherapy. Minimum follow up was five years. Methods: DNA extracted from archival malignant and non-malignant tissue was amplified by polymerase chain reaction using a panel of 11 microsatellites. MSI-H was defined as instability at greater than or equal to40% of markers, MSS as no instability, and MSI-L as instability at >0% but,40% of markers. Patients with MSI-H CRC were excluded from analysis as they have previously been shown to have better survival. Results: Thirty three MSI-L and 176 MSS CRCs were identified. There was no difference in biological characteristics or overall survival of MSI-L compared with MSS CRC but MSI-L was associated with poorer cancer specific survival (hazard ratio 2.0 (95% confidence interval 1.1-3.6)). Conclusions: Sporadic MSI-L and MSS CRCs have comparable clinicopathological features. Further studies are required to assess the impact of MSI-L on prognosis.

A microarray study of post-mortem mRNA degradation in mouse brain tissue

Background: Although there is evidence that post-mortem interval (PMI) is not a major contributor to reduced overall RNA integrity, it may differentially affect a subgroup of gene transcripts that are susceptible to PMI-related degradation. This would particularly have ramifications for microarray studies that include a broad spectrum of genes. Method: Brain tissue was removed from adult mice at 0, 6, 12, 18, 24,36 and 48 h post-mortem. RNA transcript abundance was measured by hybridising RNA from the zero time point with test RNA from each PMI time point, and differential gene expression was assessed using cDNA microarrays. Sequence and ontological analyses were performed on the group of RNA transcripts showing greater than two-fold reduction. Results: Increasing PMI was associated with decreased tissue pH and increased RNA degradation as indexed by 28S/18S ribosomal RNA ratio. Approximately 12% of mRNAs detected on the arrays displayed more than a two-fold decrease in abundance by 48 It post-mortem. An analysis of nucleotide composition provided evidence that transcripts with the AUUUA motif in the 3' untranslated region (3'UTR) were more susceptible to PMI-related RNA degradation, compared to transcripts not carrying the 3'UTR AUUUA motif. Consistent with this finding, ontological analysis showed transcription factors and elements to be over-represented in the group of transcripts susceptible to degradation. Conclusion: A subgroup of mammalian mRNA transcripts are particularly susceptible to PMI-related degradation, and as a group, they are more likely to carry the YUTR AUUUA motif. PMI should be controlled for in human and animal model post-mortem brain studies, particularly those including a broad spectrum of mRNA transcripts. (c) 2005 Elsevier B.V. All rights reserved.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

Percutaneous absorption of steroids: Determination of in vitro permeability and tissue reservoir characteristics in human skin layers

The skin localization of steroids following topical application is largely unknown. We determined the distribution of five steroids in human skin using excised epidermal, dermal, and full-thickness membranes in vitro. There was no significant difference in steroid maximum flux through epidermal and full-thickness membranes, other than significantly lower fluxes for the most polar steroid, aldosterone. Hydrocortisone had the highest dermal diffusivity and dermal penetration, and the accumulation of hydrocortisone and corticosterone was higher than that of the other steroids. Slower penetration and higher accumulation in the viable epidermis of progesterone in full-thickness skin were consistent with dermal penetration limitation effects associated with high lipophilicity. Copyright (c) 2006 S. Karger AG, Basel

In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation

Load-induced extravascular fluid flow has been postulated to play a role in mechanotransduction of physiological loads at the cellular level. Furthermore, the displaced fluid serves as a carrier for metabolites, nutrients, mineral precursors and osteotropic agents important for cellular activity. We hypothesise that load-induced fluid flow enhances the transport of these key substances, thus helping to regulate cellular activity associated with processes of functional adaptation and remodelling. To test this hypothesis, molecular tracer methods developed previously by our group were applied in vivo to observe and quantify the effects of load-induced fluid flow under four-point-bending loads. Preterminal tracer transport studies were carried out on 24 skeletally mature Sprague Dawley rats. Mechanical loading enhanced the transport of both small- and larger-molecular-mass tracers within the bony tissue of the tibial mid-diaphysis. Mechanical loading showed a highly significant effect on the number of periosteocytic spaces exhibiting tracer within the cross section of each bone. For all loading rates studied, the concentration of Procion Red tracer was consistently higher in the tibia subjected to pure bending loads than in the unloaded, contralateral tibia, Furthermore, the enhancement of transport was highly site-specific. In bones subjected to pure bending loads, a greater number of periosteocytic spaces exhibited the presence of tracer in the tension band of the cross section than in the compression band; this may reflect the higher strains induced in the tension band compared with the compression band within the mid-diaphysis of the rat tibia. Regardless of loading mode, the mean difference between the loaded side and the unloaded contralateral control side decreased with increasing loading frequency. Whether this reflects the length of exposure to the tracer or specific frequency effects cannot be determined by this set of experiments. These in vivo experimental results corroborate those of previous ex vivo and in vitro studies, Strain-related differences in tracer distribution provide support for the hypothesis that load-induced fluid flow plays a regulatory role in processes associated with functional adaptation.