Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell-specific markers and peripherin. Peripherin-positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin-positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin-positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone-specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.     

Effect of elevated K(+), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.     

Modeling of a periodic instability in paired helical filaments reveals an axial repeat

Ultrastructural studies of paired helical filaments (PHF) have been facilitated by the ability to isolate enriched fractions of detergent-insoluble forms of PHF. These fractions are composed of a relatively homogeneous population of short (usually < 400 nm) highly fragmented PHF. A small proportion of isolated PHF have highly stereotyped angled profiles that represent deformations due to structural instability. These distorted PHF can be characterized quantitatively using a simple numerical procedure that reveals that the axial instabilities occur with predictable regularity over the length of the PHF. Using a structural model of PHF, it is shown that the periodicity of the axial instability can be correlated to an axially repeated subunit of uniform size. The upper limit for the axial extent of the repeated segment was calculated to be 80 nm, similar to the size of a single one-half twist in the PHF ribbon. It is proposed that this segment may represent one type of particle in the hierarchy of structural subunits in the PHF ribbon, or an oligomeric intermediate species in PHF assembly. Received: 16 June 1999 / Revised, accepted: 7 September 1999     

Abnormal “hair-like” filaments in chronic maxillary sinusitis

Summary Scanning electron microscopic investigations were performed on the maxillary sinus mucosa of five healthy persons and seven patients with chronic maxillary sinusitis. The occurence of previously undescribed hair-like filaments in one case of chronic maxillary sinusitis is presented. These abnormal filaments are much longer (about 50–60 m) and thinner (about 0.08 m) than normal kinocilia (about 0.2 m thick and 5–7 m long), thus exhibiting a hair-like appearance. As the microvilli (cytofila) in maxillary sinus mucosa have the same thickness (about 0.08 m), these hair-like filaments could be regarded as abnormally long microvilli. The possible significance of these abnormal filaments is discussed.Supported by the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich, Projekt Nr. 3348     

培养人视网膜色素上皮细胞的免疫细胞化学特征

目的：观察体外培养人视网膜色素上皮细胞（ＲＰＥ）免疫组化特征．方法：取第１代、第３￣６代ＲＰＥ,以间接免疫荧光方法比较角蛋白、波形蛋白、肌动蛋白、ＶⅢ因子和ＧＦＡＰ,ＨＡＭ４５等６种蛋白表达的特征．结果：与对照组相比,传代培养的人ＲＰＥ角蛋白、波形蛋白表达呈阳性;随传代次数增多,角蛋白表达强度无明显变化,波形蛋白表达逐渐增强;肌动蛋白、ＶⅢ因子、神经纤维酸性蛋白（ＧＦＡＰ）、ＨＭＢ４５呈阴性．结论     

基因工程抗角蛋白抗体在正常皮肤和几种表皮增生性皮肤病皮损中的反应定位

目的　探索一株基因工程人源性抗角蛋白Fab抗体在正常皮肤及几种表皮增生性皮肤病皮损中的反应定位。方法　利用从噬菌体抗体库中筛选到的特异表达抗角蛋白Fab片段的质粒转化大肠杆菌 ,IPTG诱导表达出Fab抗体 ,纯化鉴定后用此抗体对正常皮肤及银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损进行免疫组化染色。结果　正常皮肤表皮呈阴性染色 ,毛囊呈阳性染色 ,银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损均呈现明显的阳性着色 ,其中银屑病皮损基底细胞层为强阳性。所有细胞染色部位均位于胞质 ,胞核未见着色 ,真皮为阴性。结论　该株人源性抗角蛋白Fab抗体主要与表皮组织结合 ,对银屑病等表皮增生性皮肤病的损害具有较高特异性。     

Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect.

We have previously shown that in hypothalamic mixed neuronal-glial cultures both astrocytic shape and distribution of glial fibrillary acidic protein (GFAP) are modified by estradiol. In the present study, we have investigated whether or not the presence of neurons is necessary for these hormonal effects. In mixed neuronal-glial hypothalamic cultures the proportion of process-bearing GFAP-immunoreactive cells was significantly increased after treatment for 30 min with 10(-12) M 17 beta estradiol. This effect was present for at least 1 day and was reverted by incubating the cells in estradiol-free medium. Estradiol incubation resulted in a progressive differentiation of GFAP-immunoreactive cells from a flattened epithelioid morphology to bipolar, radial, and stellate shapes. This effect was not observed in pure hypothalamic glial cultures. Furthermore, incubation of hypothalamic glial cells with medium conditioned by estradiol-treated mixed hypothalamic cultures did not affect the shape of GFAP-immunoreactive astrocytes. In contrast, addition of hypothalamic neurons, but not cerebellar neurons or fibroblasts, to established hypothalamic glial cultures affected the development of estradiol sensitivity in astrocytes. These results indicate that estradiol induction of shape changes in hypothalamic astrocytes is not only dependent on the presence of hypothalamic neurons, but that physical contact between astrocytes and neurons is necessary for the manifestation of the effect of this hormone.     

The formation of wrinkles caused by transition of keratin intermediate filaments after repetitive UVB exposure

It has been reported that the formation of wrinkles involves changes in the elastic properties of the dermis due to the denaturation of elastic fibers. Several studies have shown that the hydration condition of the stratum corneum is also important in wrinkle formation. It is, however, still unclear how the stratum corneum contributes to wrinkle formation. Here we investigated the relationship between the formation of wrinkles and changes in the physical properties and condition of the skin after repetitive ultraviolet B (UVB) irradiation of hairless mice (HR/ICR). Repetitive UVB irradiation caused wrinkles on the dorsal skin of the mice. The elasticity (E) of the stratum corneum of UVB-irradiated mice was significantly lower than that of age-matched control (unirradiated) mice. UVB exposure also caused a deterioration of the fibrous ultrastructure of keratin intermediate filaments (KIFs) in the skin. We conclude that the deterioration of KIFs in the stratum corneum caused by repetitive UVB irradiation decreases the elastic properties of the stratum corneum, resulting in the formation of wrinkles.     

The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system

It is known that two cytoskeleton components, microtubules and actins filaments, are required for efficient endocytosis. The relative importance of these two components in the cellular uptake of 2-(dimethylamino)ethyl methacrylate (DMAEMA)-DNA polyplexes was investigated in this study by applying microtubule depolymerising agent, colchicine, and actin polymerising inhibitor, cytochalasin D, in a cell transfection study. The effect of colchicine on transfection efficiency of polyplexes was found to be a time-dependent phenomenon, whereby the level of gene expression was inhibited at early stage, presumably to the disruption of a transport of vesicles along microtubules by colchicine. As time progressed, the level of gene expression was significantly enhanced relative to the control, possibly due to the failure in transport of vesicles from endosomes to late lysosomes, or due to the breakdown of nuclear membrane when mitosis was arrested at metaphase by colchicine. On the other hand, transfection efficiency was significantly reduced at all time points by cytochalasin D, which is considered to primarily affects invagination of vesicles at the early stage of endocytosis by inhibiting actin polymerisation. Further investigation to identify the endocytotic route of DMAEMA polyplexes was conducted applying clathrin- and caveolae- pathways inhibitors in cell transfection study. The results indicate that DMAEMA polyplexes were internalized primarily through clathrin-mediated pathway, with a minor fraction possibly entering cells via a caveolae-mediated pathway.