Autoradiographic demonstration of the copper-accumulating tissues in mice with a defect homologous to Menkes' Kinky Hair disease

Mutated Brindled mice have a genetic defect homologous to the human disorder Menkes' or Kinky Hair disease. These animals were used to survey the tissues in which copper accumulates as result of this defect. Heterozygous and unaffected animals were i.p. injected with radio-copper (64Cu). Sites of pathological 64Cu accumulation were visualized by whole body autoradiography. Apart from the tissues already known to accumulate copper, particularly the kidneys, pathological accumulations were also found in a number of other tissues. It is concluded that in Menkes' disease copper accumulates in epithelioid cells.     

Mutations in COA6 cause Cytochrome c Oxidase Deficiency and Neonatal Hypertrophic Cardiomyopathy

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6‐deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady‐state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse‐chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII₂/CIV_n‐supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6‐deficient patients is provided.     

Cerebellar Expression of Copper Chaperone for Superoxide,Cytosolic Cu/Zn-Superoxide Dismutase, 4-Hydroxy-2-Nonenal,Acrolein and Heat Shock Protein 32 in Patients with Menkes Kinky Hair Disease: Immunohistochemical Study

Background

To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death.

Methods

Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32).

Results

The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively.

Conclusion

In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism.     

Decreased Cytochrome c Oxidase Subunit VIIa in Aged Rat Heart Mitochondria: Immunocytochemistry

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24‐month old (aged) rats compared to 6‐month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi‐quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

The Effect of Folate Deficiency on Neuronal RNA Content: A Quantitative Cytochemical Study

One-day-old chicks were fed a defined ration deficient in folic acid. They were killed at 4 weeks of age when they showed characteristic clinical signs of folate deficiency and extremely low whole blood folate levels. Cerebellar Purkinje cells were dissected out and their total ribonucleic acid (RNA) content was determined by Edström''s microchemical technique. The total RNA content of Purkinje cells of the folate deficient chicks was significantly lower than that of control chicks fed a complete ration. The low RNA values of the folate deficient chicks were apparently not only secondary to anaemia or growth retardation, and suggest that severe folate deficiency may directly interfere with neuronal RNA synthesis. The significance of the findings is discussed with reference to human pathology.     

Involvement of Src-Suppressed C Kinase Substrate in Neuronal Death Caused by the Lipopolysaccharide-Induced Reactive Astrogliosis

Src-suppressed C kinase substrate (SSeCKS), a protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein, regulating the inflammatory process. In the process of spinal inflammatory diseases by LPS intraspinal injection, expression of SSeCKS in the spinal cord was increased, mainly in active astrocytes and neurons. Induced SSeCKS was colabeled with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (an apoptosis maker) in the late inflammation processes. These results indicated that SSeCKS might correlate with the inflammatory reaction and late neurodegeneration after LPS injection. A cell type-specific action for SSeCKS was further studied within C6 cells and PC12 cells. Knockdown of SSeCKS by small-interfering RNAs (siRNAs) blocked the LPS-induced inducible nitric oxide synthase (iNOS) expression in C6 cells, while overexpression SSeCKS enhanced iNOS expression. SSeCKS is also participated in regulation of PC12 cell viability. Loss of SSeCKS rescued PC12 cell viability, and excessive SSeCKS exacerbated the cell death upon conditioned medium and tumor necrosis factor-alpha exposure. This study delineates that SSeCKS may be important for host defenses in spinal inflammation and suggests a valuable molecular mechanism by which astrocytes modify neuronal viability during pathological states.     

肺癌组织神经内分泌分化的免疫组化研究

目的了解非小细胞肺癌(NSCLC)的神经内分泌(NE)分化特征.方法采用SP超敏免疫组化法对120例NSCLC和24例SCLC进行了神经烯醇化酶(NSE)、嗜铬粒素A(CgA)和突触素(SYN)的检测.并与各型肺癌的分型及分化程度进行了相关分析.结果三种神经内分泌标记物均为胞浆染色.NSE和SYN的阳性染色部分为弥漫性,部分为灶性分布.而CgA均为小灶性分布.在NSCLC中CgA和SYN阳性的病例NSE均阳性,除2例外CgA阳性的SYN均阳性.NSE的阳性染色除分布癌细胞外,部分间质亦可见非特异染色.在120例NSCLC中,52.5%表达NSE,9.2%表达CgA,28.3%表达SYN.在SCLC中100%表达NSE,33.3%表达CgA,54.2%表达SYN.三种标记物的阳性率与NSCLC的分型及分化程度、病人的年龄及性别均无明显关系(p>0.05).结论NSE敏感性较高,特异性较低,而CgA和SYN特异性较高,敏感性较低,故在NSCLC的临床诊断NE分化时,须三者结合应用.     

Mitochondrial D-loop and Cytochrome Oxidase C subunit I polymorphisms among the breast cancer patients of Mizoram,Northeast India

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations as it is prone to oxidative stress. The aim of the present study is to assess the novel mutations in mitochondrial genes from blood samples among the breast cancer patients from a less studied Northeast Indian population. D, B, L haplogroups were observed in the cancer samples and a total of 44 mtDNA D-loop sequence variations at 42 distinct nucleotide positions were found. All the sequence variations were transitional substitutions and 6 were heteroplasmic states, except for a cytosine copy number change (9C/8C) at np 303e309 in three samples examined. A total of 88 Cytochrome Oxidase C subunit I (COXI) sequence differences with respect to the Revised Cambridge Reference Sequence (rCRS) were identified including 20 missense variants with 100 % sample mutation frequency. All 20 missense mutations are highly conserved with a Cumulate Index of 100 %. Among 88 COXI mutations, 24 (13 were Non-Synonymous and 11 were Synonymous) were not previously reported (novel mutation) in the literature or the public mtDNA mutation databases. Analysis of three-dimensional structure of COXI open reading frame (ORF) predicted the effect of one single codon (96R > C, 217T > I, 224-225GG > EE and 227D > T) mutations located in the signal peptide binding position. Analysis of mitochondrial DNA mutations, as a viable alternative, has the advantage of being capable of detecting inherent risk factors for breast cancer development.     

A Comparative Immunohistochemical Study of Splenic Arterial Hyalinosis in Health and Disease

Hyaline deposits in arterioles and arteries of spleen were studied immunohistochemically. Hyaline lesions in arteriosclerotic heart disease were characterized by significant deposits of IgG, IgM, β1C-β 1A-globulins and β-lipoproteins. These corresponded to histochemically stained deposits of acid mucopolysaccharides and microscopic areas of musculoelastic tissue damage in the hyaline masses. While, in young adults and a few other cases of other diseases, an occasional granular to linear deposit of IgG, IgM, β1C-β 1A-globulin and β-lipoprotein was noted, no localization of IgA, rabbit antihuman fibrin and rabbit antihuman fibrinogen was seen. A variety of other histochemical staining reactions were found to be negative. These findings suggest that: a) hyaline deposits in splenic arterioles and arteries occur with greater severity in patients with hypertensive and arteriosclerotic heart disease; b) a possible abnormality related to filtration defects in arteries and arterioles, resulting in the trapping of plasma proteins, appears likely; c) increased localization of acid mucopolysaccharides and destruction of musculoelastic tissue is not an uncommon feature in hyaline masses; d) fibrin is not a component of these deposits and e) further study of other organs is necesary to observe the composition of hyaline in arterioles and arteries.     

Microvasculature in Brain Biopsy Specimens from Patients with Alzheimer's Disease: An Immunohistochemical and Ultrastructural Study

Brain biopsy specimens from five patients with Alzheimer's disease obtained in the course of a trial of intracerebroventricular bethanechol were studied by immunohistochemical (antibody to A₄ peptide) and ultrastructural techniques, with particular emphasis on the microvessels. In some cases, numbers of A₄-immunoreactive lesions (senile plaques) correlated well with numbers of plaques demonstrable by silver stains. Prominent A₄-immunoreactive amyloid angiopathy was seen in one patient. The patient with severe cerebral amyloid angiopathy (CAA) showed extensive arteriolar deposition of amyloid filaments with apparent destruction of the media but remarkably intact endothelium. A cell of origin for amyloid filaments was not apparent, although close proximity to smooth muscle cell remnants in the arteriolar media suggested this as one possible cell of origin. Frequent vessels showed medial or adventitial collagen deposition, even when the amount of amyloid was minimal or negligible. Thus relatively severe CAA can exist in the absence of overt endothelial injury, although related studies on this tissue indicate definite abnormalities of the blood-brain barrier. Conversely, destruction of smooth muscle cells and collagen deposition in vessel walls may be the cellular correlates of arteriolar weakening that can lead to CAA-related brain hemorrhage.     

Microvasculature in Brain Biopsy Specimens from Patients with Alzheimer's Disease: An Immunohistochemical and Ultrastructural Study

Brain biopsy specimens from five patients with Alzheimer's disease obtained in the course of a trial of intracerebroventricular bethanechol were studied by immunohistochemical (antibody to A₄ peptide) and ultrastructural techniques, with particular emphasis on the microvessels. In some cases, numbers of A₄-immunoreactive lesions (senile plaques) correlated well with numbers of plaques demonstrable by silver stains. Prominent A₄-immunoreactive amyloid angiopathy was seen in one patient. The patient with severe cerebral amyloid angiopathy (CAA) showed extensive arteriolar deposition of amyloid filaments with apparent destruction of the media but remarkably intact endothelium. A cell of origin for amyloid filaments was not apparent, although close proximity to smooth muscle cell remnants in the arteriolar media suggested this as one possible cell of origin. Frequent vessels showed medial or adventitial collagen deposition, even when the amount of amyloid was minimal or negligible. Thus relatively severe CAA can exist in the absence of overt endothelial injury, although related studies on this tissue indicate definite abnormalities of the blood-brain barrier. Conversely, destruction of smooth muscle cells and collagen deposition in vessel walls may be the cellular correlates of arteriolar weakening that can lead to CAA-related brain hemorrhage.     

Ultrastructural Study of Renal Involvement in Two Females with Anderson-Fabry Disease

Anderson-Fabry disease (AFD) is a rare X-linked lipid storage disorder due to a deficient lysosomal α-galactosidase A (α-Gal) activity. In males with the classic form of the disease the enzymatic defect leads to progressive accumulation of glycosphingolipids (GL) in different organs, mainly in the kidney, heart, and brain, causing severe multisystem failure. AFD is usually mild in heterozygous females, but severe cerebrovascular, renal, and cardiac manifestations have been rarely described. The aim of this study is to describe renal involvement of mild symptomatic female carriers by ultrastructural analysis focusing to microvascular lesions, considered to be one of the major causes of systemic disease in AFD. Resin-embedded renal biopsies from 2 sisters with isolated mild proteinuria and belonging to a family group with AFD were observed by light and electron microscopy. In spite of the mild clinical symptoms, diffuse GL storages were demonstrated in all types of glomerular cells and in interstitial endothelial cells. Moreover, platelets were frequently observed in glomerular vassels, a feature coherent with a possible role of prothrombotic state, and platelet activation, in early glomerular lesions.     

Neuronal and glial localization of guanylate cyclase. Immunohistochemical evidence in cultured cells

A study has been carried out on the localization of guanylate cyclase employing cultured brain cells. Guanylate cyclase has been found to be located in neurons as well as in glial cells. This has been supported by the immunohistochemical as well as biochemical data.