Respective Control Tissues Research Articles

Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1): a target for cancer therapeutic intervention?

BackgroundCap-dependent mRNA translation is essential for the translation of key oncogenic proteins at optimal levels and is highly regulated by the rate limiting, initiation step in protein synthesis. Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) serves as a scaffold for assembly of cap-dependent translation components in EIF4F complex formation. In the current study, we analyzed the role and expression of EIF4G1 in Pan human cancer panels through various approaches.MethodsImmunohistochemistry analysis of EIF4G1 protein was done on high-density multi-organ Human Cancer tissue microarray (TMA) derived from the patient samples from different cancers. We used multiple clinical cohorts to analyze the EIF4G1 mRNA expression across human cancers. TCGA data analysis of EIF4G1 was done through Ualcan and c-bioportal web servers. Western blots for EIF4G1 protein was done for different cell lines in representing the multiple cancer types. Dependency score was calculated through Cancer Dependency Map. Clonogenic, tumorosphere assay and cell invasion assay were done with EIF4G complex inhibitor. Association of EIF4G1 mRNA and Kaplan–Meier survival analysis was done on available TCGA datasets.ResultsWe observed an increase in EIF4G1 protein levels in tissue sections from different cancers as compared to their respective normal tissue. Our analysis of the TCGA data revealed that EIF4G1 mRNA expression is significantly increased in tumor tissues compared to respective control tissues across human cancers and variable expression was observed among different datasets. We discovered that alteration frequency in EIF4G1 is prevalent in human cancers e.g. prostate cancer (~ 25%), ovarian cancer (~ 15%), Head and Neck cancer (~ 13%) and cervical cancer (~ 12.5%). EIF4G1 mRNA and protein levels were high across cancer cell lines from multiple organs. Our analysis of DepMap datasets utilizing depletion assays revealed that EIF4G1 is critical for cancer cell survival. Treatment with EIF4G complex inhibitor impaired clonogenic, tumorosphere formation potential and inhibited cell invasion. Moreover, higher EIF4G1 mRNA level was associated with a lower median survival of patients in multiple tumor types.ConclusionsThese studies show that EIF4G1 is amplified/over-expressed in multiple cancers and plays an essential role in cancer cell survival, as such EIF4G1 could serve as a novel potential target for therapeutic intervention across many cancers.

An iOS App to Expedite the Evaluation of Immunohistochemistry Control Tissue.

Control tissues play a vital role in diagnostic immunohistochemistry. They serve to document that the appropriate antibody was used, on the correct control tissue, and run on optimized conditions. As part of the evolving process of standardization in diagnostic immunohistochemistry, specific tissues have been identified based on agreement by experts in this field capable of serving as the benchmark(s) for several antibodies. These tissues are recommended based on known and predictable levels of strong, weak, and no expression of the antigen being queried. These tissues can be used for positive and negative control purposes, respectively, and are regarded as primary positive and/or negative external control tissue. If some of these tissues are not present in sufficient numbers in a laboratory's archive for daily use, they can still be used as the basis to evaluate other tissues that are not as well characterized and chosen to serve as secondary and external positive controls. In this manner, either the former or latter can function as external positive and negative control tissue for the quality assurance of immunohistochemistry done in a laboratory. The use of the selected tissues may be applicable to the detection of several different antigens by a number of separate antibodies, with differences in the staining of specific cells or the localization in staining within those cells. However, the amount of information needed to be familiar with to render a correct interpretation of the control tissue may prove daunting. One means of dealing with this problem would be to create a document capable of serving as a reference guide. Traditional types of references, however, may suffer from issues related to convenience, updating, and mobility. Herein we describe a mobile device application (app) created to serve as a reference for control block tissues. This app can capably house and easily retrieve all the relevant information on all the antibodies and their respective control tissues in a laboratories test menu, thus enabling the use of standardized tissues as control material and spreading the ability to perform immunohistochemical quality control to individual pathologists.

Inactivation of the LKB1-AMPK signaling pathway does not contribute to salivary gland tumor development - a short report.

Activation of AMPK by the tumor suppressor LKB1 represents an essential gatekeeping step for cells under energetic stress to prevent their growth and proliferation by inhibiting mTOR activation, until the energy supply normalizes. The LKB1/AMPK pathway is frequently downregulated in various types of cancer, thereby uncoupling tumor cell growth and proliferation from energy supply. As yet, little information is available on the role of the LKB1/AMPK pathway in tumors derived from salivary gland tissues. We performed LKB1 protein expression and AMPK and mTOR activation analyses in several salivary gland tumor types and their respective healthy control tissues using immunohistochemistry. No significant downregulation of LKB1 expression or decreased activation of AMPK or mTOR were observed in any of the salivary gland tumors tested. In contrast, we found that the salivary gland tumors exhibited an increased rather than a decreased AMPK activation. Although the PI3K/Akt pathway was found to be activated in most of the analyzed tumor samples, the unchanged robust activity of LKB1/AMPK likely prevents (over)activation of mTOR. In contrast to many other types of cancer, inactivation or downregulation of the LKB1/AMPK pathway does not substantially contribute to the pathogenesis of salivary gland tumors.

Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes

BackgroundAs on today, the global mortality rate of gallbladder cancer is still very high. Both genetic and epigenetic alterations play pivotal roles in the development of cancer. We selected seven tumour associated genes, implicated in other cancers, to assess their methylation status in gallbladder cancer and gallstone diseases. Aim of studyTo study the promoter methylation of certain tumour associated genes in the molecular pathogenesis of gallbladder cancer and gall stone diseases. Materials and methodsMethylation specific PCR for seven tumour associated genes, viz., MASPIN, 14-3-3 sigma gene, THBS1, FLNC, HLTF, COX-2 and SOCS1, was performed in 50 gallbladder cancer (GBC), 30 gall stone diseases (GSD) and their respective adjacent control tissues. Semi-quantitative PCR and immunohistochemistry was carried out to check the expression level. Student's t-test was carried out to compare the differences in the methylation and expression patterns between cases and control tissues. ResultsWe observed methylation of CpG islands in seven of the studied markers, but, the frequency of methylation was found varying among different samples. Of them, 14–33 sigma showed methylation in 45 GBC (90%; p=0.0001) and 25 GSD (86.66%; p=0.001), MASPIN in 35 GBC (70%; p=0.0008) and 18 GSD (51.43%; p=0.040), FLNC in 16 GBC (32%; p=0.0044) and 9 GSD (25.71%; p=ns), THBS1 in 26 GBC (52%; p=0.0009) and 10 GSD (28.57%; p=0.0505), HLTF in 8 GBC (16%; p=ns) and 2 GSD (5.71%; p=ns), COX2 in 10 GBC (20%; p=ns) and 6 GSD (17.14%; p=ns) and SOCS-1 in 3 GBC samples only (6%; p=ns), but not in GSD. Semi-quantitative PCR revealed down regulation in MASPIN, 14-3-3 sigma, THBS1, HLTF, COX2 and SOCS1 in advanced gallbladder cases. Immunohistochemistry further confirmed the down-regulation of SOCS1 in GBC. ConclusionThe present study infers that accumulation of epigenetic alterations increases poor prognosis of GBC patients. Out of seven genes, MASPIN and THBS1 play key epigenetic role in GBC, but not in GSD. The reason for downregulation of SOCS1 only in GBC, and unaltered expression of 14-3-3 sigma protein in all the GBC and GSD tissue samples is not clear. Further investigation on the expression pattern of these genes in GBC cell lines may elucidate their likely functional role in in association with gallbladder cancer.

DDX39 acts as a suppressor of invasion for bladder cancer

The object of the present study was to identify markers for predicting urinary bladder cancer progression by comparative proteome analysis of bladder cancers and paired normal mucosas. We found that DDX39 was overexpressed in four of six bladder cancers examined compared with respective control tissues. Immunohistochemical analysis using 303 bladder cancer specimens revealed that DDX39 was inversely correlated to pT stage and histological grade progression. The incidence of DDX39(high) tumors (positive cells ≥50%) was 68.6%, 43.5%, 20.0%, and 5.3% in pTa, pT1, pTis, and ≥pT2 tumors, respectively, and 65.2%, 60.7%, and 19.6% in G1, G2, and G3 tumors, respectively. The incidence of DDX39(high) tumors was significantly lower in pT1 and ≥pT2 compared to pTa tumors, and also significantly lower in G3 compared to G1 and G2 tumors. Follow-up analysis (n = 105) revealed that DDX39(low) tumors (positive cells <50%) were associated with disease progression (hazard ratio 7.485; P = 0.0083). Furthermore, DDX39-knockdown bladder cancer cells increased their invasion ability compared to negative control cells. These results suggest that DDX39 is a suppressor of invasion and loss of its function predicts disease progression in bladder cancers.

Global expression profiling of sex cord stromal tumors fromMen1heterozygous mice identifies altered TGF‐β signaling, decreased Gata6 and increased Csf1r expression

Heterozygous disruption of the Men1 gene predisposes mice to the development of multiple endocrine tumors, accurately mimicking the human MEN1 cancer predisposition syndrome. Additionally, Men1(+/-) mice frequently develop sex cord adenomas. The mechanism underlying the susceptibility of these mice to sex cord tumor development has not been fully determined, but data suggest it may involve transcriptional regulation of key growth promoting/repressing genes. To identify potential menin-regulated genes that may be important for tumor suppression in sex cord cells, we compared the global gene expression profiles of testis and ovary adenomas with other endocrine tumors of the pancreas and pituitary from Men1 heterozygous mice and with control tissues. Gonadal tumors clustered separately from pancreas and pituitary tumors with only a few genes (e.g., Cdkn2c) commonly dysregulated in all tumor types. Testis and ovary tumors displayed a higher level of transcriptional similarity to each other than they did to their respective control tissues. Among genes that had decreased expression in tumors was significant over-representation of genes associated with the TGF-beta, hedgehog and Wnt signaling, indicating that loss of menin function affects these pathways at the level of transcription. Aberrant protein expression in Leydig and granulosa cells of 2 transcriptionally dysregulated gene products, Gata6 and Csf1r were confirmed by immunohistochemistry. We propose that sex cord tumor susceptibility in Men1(+/-) mice involves deregulated cell proliferation due to dysregulation of multiple cell growth regulating genes including: reduced Cdkn2c transcription, loss of TGF-beta pathway tumor suppressor function (e.g., Gata6) and transcriptional activation of Csf1r.

Overexpression of E2F1 in Clear Cell Renal Cell Carcinoma: A Potential Impact of Erroneous Regulation by Thyroid Hormone Nuclear Receptors

We show here that the promoter of E2F1 gene, encoding one of the key regulators of cell proliferation, is overly active in the presence of low amounts of triiodothyronine (T3) and in the presence of mutant thyroid hormone receptor. We also show that T3-thyroid hormone receptor pathway of regulation of molecular processes is disturbed in clear cell renal cell carcinoma (ccRCC) on several levels, including overexpression of thyroid hormone receptors and the disturbance of their binding to DNA and to the hormone. In comparison to the cancer-free kidneys and peritumoral respective control tissues, E2F1 mRNA and protein levels are significantly increased in cancer tissues. A significant correlation between E2F1 mRNA and protein levels has been found in both control types and ccRCCs. No correlation was observed between the amount of E2F1 mRNA and the amount of thyroid hormone receptors or their DNA or T3 binding activity, suggesting that the function of thyroid hormone receptors could be markedly disturbed in both tumor and peritumoral cells. In summary, we show that ccRCC is characterized by the overexpression of E2F1, which is likely a result of a deregulated control of T3-dependent molecular processes.

Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy

BackgroundTransient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established.MethodsWe have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods.ResultsTRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons.ConclusionThe accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.

Dietary modulation of fatty acid profiles and oxidative status of rat hepatocyte nodules: Effect of different n−6/n−3 fatty acid ratios

Male Fischer rats were fed the AIN 76A diet containing varying n-6/n-3 FA ratios using sunflower oil (SFO), soybean oil (SOY), and SFO supplemented with EPA-50 and GLA-80 (GLA) as fat sources. Hepatocyte nodules, induced using diethylnitrosamine followed by 2-acetylaminofluorene/partial hepatectomy promotion, were harvested, with surrounding and respective dietary control tissues, 3 mon after partial hepatectomy. The altered growth pattern of hepatocyte nodules in rats fed SFO is associated with a distinct lipid pattern entailing an increased concentration of PE, resulting in increased levels of 20:4n-6. In addition, there is an accumulation of 18:1 n-9 and 18:2n-6 and a decrease in the end products of the n-3 metabolic pathway in PC, suggesting a dysfunctional delta-6-desaturase enzyme. The hepatocyte nodules of the SFO-fed rats exhibited a significantly reduced lipid peroxidation level that was associated with an increase in the glutathione (GSH) concentration. The low n-6/n-3 FA ratio diets significantly decreased 20:4n-6 in PC and PE phospholipid fractions with a concomitant increase in 20:5n-3, 22:5n-3, and 22:6n-3. The resultant changes in the 20:4/20:5 FA ratio and the 20:3n-6 FA level in the case of the GLA diet suggest a reduction of prostaglandin synthesis of the 2-series. The GLA diet also counteracted the increased level of 20:4n-6 in PE by equalizing the nodule/surrounding ratio. The low n-6/n-3 ratio diets significantly increased lipid peroxidation levels in hepatocyte nodules, mimicking the level in the surrounding and control tissue while GSH was decreased. An increase in n-3 FA levels and oxidative status resulted in a reduction in the number of glutathione-S-transferase positive foci in the liver of the GLA-fed rats. Modulation of cancer development with low n-6/n-3 ratio diets containing specific dietary FA could be a promising tool in cancer intervention in the liver.

High b-value q-space analyzed diffusion-weighted MRI: application to multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) which affects nearly one million people worldwide, leading to a progressive decline of motor and sensory functions, and permanent disability. High b-value diffusion-weighted MR images (b of up to 14000 s/mm(2)) were acquired from the brains of controls and MS patients. These diffusion MR images, in which signal decay is not monoexponential, were analyzed using the q-space approach that emphasizes the diffusion characteristics of the slow-diffusing component. From this analysis, displacement and probability maps were constructed. The computed q-space analyzed MR images that were compared with conventional T(1), T(2) (fluid attenuated inversion recovery (FLAIR)), and diffusion tensor imaging (DTI) images were found to be sensitive to the pathophysiological state of white matter. The indices used to construct this q-space analyzed MR maps, provided a pronounced differentiation between normal tissue and tissues classified as MS plaques by the FLAIR images. More importantly, a pronounced differentiation was also observed between tissues classified by the FLAIR MR images as normal-appearing white matter (NAWM) in the MS brains, which are known to be abnormal, and the respective control tissues. The potential diagnostic capacity of high b-value diffusion q-space analyzed MR images is discussed, and experimental data that explains the consequences of using the q-space approach once the short pulse gradient approximation is violated are presented.

Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta

A diet containing 0.3% cholesterol was given to male New Zealand rabbits for 16 weeks; this produced atherosclerotic lesions (fatty streaks) on 80% of the intimai surface of the thoracic aorta and on 45% of the intimai surface of the abdominal aorta. The endothelium-dependent relaxations induced by acetylcholine, substance P and ionophore A23187 were inhibited in the atherosclerotic aortas. Besides the endothelium-independent relaxations induced by nitroglycerine, the relaxations induced by atrial natriuretic peptide (ANF) were also significantly reduced in the more atherosclerotic thoracic aorta. In bioassay experiments it was found that acetylcholine and substance P caused a smaller release of endothelium-derived relaxing factor (EDRF) from atherosclerotic thoracic aortas than from control thoracic aortas: the EDRF released by the vasodilators evoked less relaxation in atherosclerotic detector abdominal aortas than in control detector abdominal aortas. Nitric oxide evoked significantly less transient relaxation in the atherosclerotic thoracic and abdominal aortas than in the respective control tissues. The data indicate that as experimental atherosclerosis in the rabbit progresses, both vascular activity and EDRF release become affected; this leads to a complete loss of endothelium-dependent relaxation in the more atherosclerotic blood vessels.

Acinar cell carcinoma of rat pancreas: regulation of cholesterol esterification.

The regulation of cholesterol esterification during cell proliferation was studied. The serum free cholesterol, cholesterol esters and lecithin: cholesterol acyltransferase (LCAT) activity of nude mice with and without pancreatic acinar cell tumours and rats with proliferating tissues were determined. In addition, the apparent activity of acyl-CoA: cholesterol acyltransferase (ACAT) in homogenates of nude mouse tumours and proliferating rat tissues were determined and compared with those of normal nude mouse and rat tissues. Serum cholesterol ester levels were significantly lower in host nude mice with tumours and in rats with regenerating liver, and increased significantly in pregnant rats when compared with respective controls. Circulating LCAT activity levels decreased in host nude mice, in pregnant rats, and in rats with regenerating pancreas and regenerating liver. Apparent ACAT activity levels increased significantly in nude mouse tumours and in foetal and postnatal rat pancreata and also in postnatal liver. At the same time, apparent ACAT activity levels decreased in foetal and regenerating rat livers when compared with respective control tissues. These results suggest that serum cholesterol esters, circulating LCAT and cellular ACAT levels are modulated during cell proliferation.

Accumulation of norepinephrine by tissue slices from spontaneously hypertensive rats.

Accumulation of 3H-1-norepinephrine (3H-1-NE) by heart and spleen slices from different aged spontaneously hypertensive rats (SHR) was compared with accumulation by slices from comparably aged normotensive rats (Wistar-Kyoto, WKY). Between weeks 4 and 9 there was a significant increase in accumulation by heart slices of the WKY; there was no further increase at weeks 13 and 16. The developmental pattern in slices of WKY spleen was different. Accumulation was the same at weeks 4 and 9; subsequently, there were significant increases at weeks 13 and 16. Slices of heart from SHR showed the same pattern of development as those from WKY. However, accumulation by slices of SHR spleen was significantly different from that by slices of WKY spleen. At all ages studied, SHR slices accumulated less 3H-1-NE. Furthermore, there was no significant increase in accumulation with age. The data show: 1. the course of development of the adrenergic neuron transport system is different in heart and spleen, and 2. accumulation of 3H-1-NE by SHR spleen, but not heart slices, is different from accumulation by the respective control tissues.

Lipid-chemical features of human gastric cancerous, polypous and ulcerative tissues.

Fourteen cases of gastric cancer, 9 cases of gastric polyp and 4 cases of gastric ulcer were studied on their tissues lipid-chemically with the aim to clarify the biochemical differences between malignant and benign growth of the human gastric tissue. Tissues were collected by surgical operation or a biopsy together with the normal tissues surrounding the lesions. One part of each tissue was subjected to histologic examination and the other part to lipid analyses by means of TLC and GLC. The total lipid content was dcreased in 71% of gastric cancerous cases and in 75% of both cases of gastric polyp and gastric ulcer as compared with the respective control tissues. The phospholipid fatty acid content of the lesions was decreased in 83% of cancerous cases but in 50% of polypous cases as compared with the respective control tissues. As for the phospholipid fatty acid composition, the percentage value of C20:4 was increased and the percentage value of C18:2 was decreased in gastric cancerous tissues when compared with those of gastric polypous tissues. Such a change of the phospholipid fatty acid composition may produce the change of the fluidity of the cell membrane and may provoke a malignant growth of human gastric cancer cells.

Further studies on lipid-chemical differences between cancerous and adenomatous polypous tissues in the human large intestine.

17 cases of cancer and 18 cases of adenomatous polyp in the large intestine were subjected to lipid-chemical studies of their tissues in order to clarify the biochemical differences between malignant neoplastic growth and benign growth. Cancerous and adenomatous polypous tissues were collected by biopsy or surgical operation together with the respective control tissues. One part of the total lipid extracted from each tissue was separated into triglyceride and phospholipid fraction by a thin-layer chromatography (TLC). The fatty acid composition and content of each lipid fraction were measured by a gas-liquid chromatography (GLC). When the fatty acid composition of a phospholipid was expressed in terms of the deviation rate, that of C14:0 was remarkably increased and that of C20:4 was remarkably decreased in the cancerous cases as compared with the respective values of the cases of adenomatous polyp. More definite differences were recognized between the cancerous cases and the cases of adenomatous polyp, when the ratio of the deviation rate of C14:0 to that of C20:4 was calculated in each case. Namely, the ratios distributed between 2.064 and 4.125 in 10 cancerous cases, and between 0.393 and 1.856 in 16 cases of adenomatous polyp, indicating the biochemical differences between cancerous tissues and adenomatous polypous tissues.

Analysis of fluid in capsules implanted into dog brain.

Perforated capsules were implanted aseptically into the brain parenchyma of dogs. The tissue and fluid formed within these intracerebral capsules (BC) were analysed for their ions and protein content. The results were compared with those for other perforated capsules implanted simultaneously into the subcutaneous tissue and muscle, respectively (denoted SC and MC). The following results were obtained:1) In BC and MC, the Na/K ratio was different from that of the respective control tissues (cerebral parenchyma and temporal muscle) but similar to that of both the tissue in SC and control connective tissue. The water content of the three different capsular tissues was almost identical, but in each case different from that of the respective control tissues.2) The Na/K ratio, Cl and protein content, and electrophoretic patterns of the three capsular fluids were similar to one another, and to serum. However, all (including BC fluid) were different in composition from the CSF (cerebrospinal fluid).3) Investigations of the effect of time lapse after capsular implantation showed that the Na and K content was generally constant over the observed period, except that the K value was high in the initial few weeks.4) Based on the results obtained, and on our data for pressure reequilibration in BC, the nature of the fluid compartment in BC is discussed. It is considered to represent an extension of the narrow space existing physiologically between the blood capillary wall and glial membrane, and not to be part of the true interstitial fluid compartment.5) The barrier existing between the blood and brain parenchyma may be regarded not as a single membrane but as two membranes of dissimilar nature.

Otosclerosis. A further investigation of inorganic constituents by neutron activation analysis.

The levels of isotopes of calcium, iodine, magnesium, copper, chlorine, and sodium in stapedes and cortical bone from otosclerotic and non-otosclerotic patients were deternined by neutron activation analysis. No n significant differences were found in the levels of calcium, iodine, magnesium, chlorine, or sodium in otosclerotic stapedes or cortical bone when compared with the levels in the respective control tissues. Simi- s larly, no difference was found between copper levels s in otosclerotic and normal stapedes.