Bruch's membrane of the brachymorphic mouse

Bruch's membrane exists between the retinal pigment epithelium and the choriocapillary endothelium. Its structure is very complicated, having five sublayers containing basement membranes of retinal pigment epithelium and choriocapillary endothelium, outer and inner collagenous layers, and a central elastic layer. In the development of Bruch's membrane in normal mice, both basement membranes are created first. Secondarily, collagen fibers are accumulated in the space between these basement membranes and then form a collagenous layer. Finally, the elastic layer elaborated in the collagenous layer separates this into outer and inner collagenous layers. Brachymorphic mice have a disorder in the sulfation pathway, resulting in undersulfation. Consequently, in Bruch's membrane of brachymorphic mice, the expression of decorin, a small proteoglycan containing chondroitin sulfate and an indispensable component in collagen assembly, is at a very low level. It is clear that hypoplasia of the collagenous layer in Bruch's membrane of brachymorphic mice induces a disorder in the following formation of the elastic layer. These findings suggest that the formation of the collagenous layer, regulated with acidic glycoconjugates such as decorin, is important in the development of Bruch's membrane.     

用膜亲和层析法纯化小鼠腹水中的单克隆抗体

目的 用膜亲和层析法（MAC）纯化小鼠腹水中单克隆抗体（mAb)。方法 采用尼龙滤膜ZBM作为介质吸附抗人血清白蛋白（HSA）,将含mAb的腹水滤过此ZBM,再将吸附于ZBM上的mAb解离下来,获得纯化的mAb。结果 直径50mm吸附了HSA的ZＢＭ。经１０mL腹水循环滤过两次,即可达到最大mAb结合量。从ＺＢＭ上解离mAb,仅需解离液滤过１次就可完成,纯化的mAb经ＰＡＧＥ检测,只显示１条带。用纯化的mAb做金标记斑点免疫渗滤法（ＤＩＧＦＡ）检测ＨＳＡ标准品时,其灵敏度较用未纯化的腹水时提高了２０倍,结论 用尼龙滤膜ＺＢＭ改进的膜亲和层析法,可用于纯化中的单克隆抗体 且简便、省时、有效。     

Apoptosis-related changes in plasma membrane glycoconjugates of peripheral blood lymphocytes in rheumatoid arthritis

Control of apoptosis (apo) is very important for diagnosis, prognosis and treatment of rheumatoid arthritis (RA). Recently, we found that an appearance of specific cell surface GC is attributable to apo and developed lectin-induced agglutination test for apo evaluation. The aim of current study was to estimate changes in surface GC expression in peripheral blood lymphocytes (PBL) of normal healthy donors (NHD) and patients with RA, measured by cell agglutination with the galactose-specific VAA lectin and by lectin-cytochemical analysis. In parallel, these changes in apo incidence were evaluated by the detection of cells with sub-G1 DNA content. The data revealed an increased level of apo in lymphocytes of RA patients (n = 29), and increased cell surface GC expression in lymphocytes of NHD (n = 18). A correlation (R = 0.708) was observed between these two indicators. Specific changes in cell surface GC can be effectively used for the detection of apo cells in RA and other autoimmune disorders.     

Propanediol-induced alterations in membrane integrity, metabolism and developmental potential of mouse zygotes

The effects of 1, 2-propanediol (PROH) on embryonic development,membrane integrity and metabolism on B₆D₂F₁ mouse zygotes inthe pronuclear stage were evaluated. In both the control andthe group treated with 1.5 M PROH, 78% of the zygotes developedinto 2-cell embryos. With 3 M PROH, the proportion of 2-cellembryos was only 7% (P < 0.05). In a second series of experiments,pronuclear mouse eggs were incubated in either fluorescein diacetate(FDA) or 5µM Acridine Orange (AO) then transferred toPROH. FDA-induced fluorescence, which is maintained until thecell membrane is damaged, was retained in 100% of the controland 98% of the zygotes treated with 1.5 M PROH. Exposure to3.0 and 6.0M PROH reduced the percentage of zygotes with FDA-inducedfluorescence to 81% (P < 0.05) and 5% (P < 0.05) respectively.AO fluoresces yellow-green within the physiological pH range(7.4). After AO exposure, 95% of the control zygotes and 95%of the zygotes exposed to 1.5 M PROH possessed yellow–greenfluorescence, indicating a normal cellular pH. Treatment with3.0 and 6.0 M PROH caused a shift in the fluorescence such that93% (P < 0.05) and 100%(P < 0.05) of the zygotes respectivelyno longer fluoresced yellow-green, indicating a lower pH. Theseresults demonstrate that a 20-min exposure to 1.5 M PROH doesnot affect embryonic development, while PROH at 3.0 M inhibitsembryonic development. Treatment with 3 M PROH causes both cellmembrane damage and pH changes, which are in turn associatedwith a decrease in embryonic development.     

Peanut lectin binding to the alveolar lining layer in hyaline membrane disease

Summary The authors have studied the histochemical pattern of Peanut lectin (PNA) binding sites in lungs of seven newborns with hyaline membrane disease (HMD) and ten controls. The alveolar lining layer was positive in HMD and no changes in the PNA pattern was noted after neuraminidase digestion. Hyaline membranes were generally unstained but occasional reactivity was encountered in some parts. No reaction with PNA was observed in control lungs, but positivity was seen after neuraminidase pretreatment. Our histochemical data document the presence of accessible galactosyl residues with absence of terminal sialic acid in the alveolar lining layer of newborns with HMD. The authors suggest that PNA reactivity in HMD reflects an histochemical feature described in fetal lungs at the pseudoglandular and canalicular stages.This work was supported by grants from MPI Rome     

A simple organ culture method for differentiation of melanocytes in mouse embryonic skin using “origami” membrane filter

Summary A simple organ culture method for culturing embryonic skin was developed. A piece of skin with a part of the neural tube from mouse embryo (11 to 12 d) was placed on a 25 mm d membrane filter. The filter was folded to wrap the explant and inserted into glass tubing. The explant and filter in the glass tubing were placed in a rotating tissue culture tube containing 5 ml culture medium (Ham's F12 supplemented with 15 to 20% fetal bovine serum) and filled with a mixture of 95% air:5% CO₂. In explants cultured for 6 d fully differentiated melanocytes were observed in the epidermis.     

Development of Reichert's membrane in the early mouse embryo

Although the composition of Reichert's membrane, a thick multilayered basement membrane between the parietal endoderm cells and the trophoblast cells of rodents, has often been investigated, the site of its production remains a subject of controversial discussion. In particular, the role of the trophoblast cells is unclear. In the present work we examined the initial development of Reichert's membrane in the early mouse embryo, using glutaraldehyde fixation with tannic acid. In the early blastocyst the occurrence of a tannic-acid-positive layer located at the inner surface of the mural trophoblast indicated the onset of basement membrane formation by the trophoblast cells. In the peri-implantation phase, this basement membrane extended into lateral areas of the inner cell mass separating the newly differentiated ectoderm and endoderm cells from each other. In these lateral regions, where the recently formed primitive endoderm cells had been attached to the monolayered basement membrane of the mural trophoblast, the membrane began to reveal the typical multilayered structure of Reichert's membrane. Our findings indicate that the initial formation of Reichert's membrane begins with the formation of a basement membrane of the mural trophoblast cells, followed by an apposition of basement membrane material, probably synthesized by primitive endoderm cells, along this primary membrane.     

骨膜提取物诱导小鼠骨髓间充质干细胞分化为类成骨细胞

目的 应用骨膜提取物诱导骨髓间充质干细胞(MSCs)分化为类成骨细胞,为骨组织_丁程提供充足的种子细胞.方法 切取小鼠颅骨骨膜提取物,诱导小鼠骨髓间充质干细胞.诱导分化后的细胞采用ALP染色、RT-PCR、蛋白印迹技术进行检测.结果 形态学观察显示,分化后的MSCs细胞形态从长梭形变成三角形、多角形或立方形;ALP活性...     

Cell surface glycoconjugates and the extracellular matrix of the developing mouse embryo epicardium

Cell surface glycoconjugates and the extracellular matrix (ECM) of the proepicardium and the developing epicardium were studied in early mouse embryos by light and electron microscopy with histochaemical and immunocytochaemical techniques. The extracardially located proepicardium consists of polarized mesothelial cells forming the proepicardial vesicles. These vesicles contain a fine proteoglycan network and an acellular ECM rich in hyaluronic acid. Membrane-bound glycoconjugates are shown with cuprolinic blue, alcian blue and ruthenium red on the apical (outer) cell surface, while fibronectin and laminin are present on the basal (luminal) cell surface. These membrane and matrix components of the proepicardium might be involved in specific attachment of proepicardial cells to the bare heart tube and might facilitate the initial migration of epicardial cells over the myocardial surface. In the cell coat of the cardiomyocytes of the bare heart tube the fibronectin and laminin are concentrated in patches. The formation of the epicardial covering is a rapid process, requiring only about 2 days (9–11 days) to ensheath the entire heart tube from the inflow to the outflow segment. The subepicardial matrix between the newly formed epicardial covering and myocardial layer is acellular at first, but contains a condensing proteoglycan network, membrane and matrix fibronectin, type IV collagen and laminin on the myocardial cell surface. The formation and the distribution of the subepicardial ECM show regional characteristics. The accumulating ECM forms wide subepicardial spaces and protuberances in the atrioventricular and interventricular sulci. The sulci of the heart seem to provide the optimum microenvironment for haematopoiesis and vasculogenesis. Haematopoietic islands and coronary vessel forerunners appear and concentrate in the regularly spaced surface protuberances. The vasculogenesis proceeds from the inflow to the outflow segment of the heart. The first blood capillaries appear in the sinoatrial sulcus of the 10-day embryo. By 11–13 days the subepicardial blood vessels form an interconnected network and establish the coronary artery orifices.     

人生精细胞内酶性磷酸酶活性与膜结构关系的研究

目的　研究人生精细胞内酸性磷酸酶 (ACP)活性与细胞膜结构之间的关系。方法　选取 5例抗精抗体 (AsAb)阳性 (不育A组 ) ,9例生殖系统有过炎症中 (不育B组 )及 6例原因不明 (不育C组 )共 2 0例男性不育患者新鲜精液 ,同时取 8例正常生育男性新鲜精液作对照 ,用Gomori s铅法对标本进行孵育后 ,在透射电镜下观察。结果　AsAb阳性和有炎症史男性不育患者标本中 ,见较多的脱落生精细胞 ,有大量与铅结合的ACP显现于溶酶体 ,ACP活性明显升高。ACP阳性反应数在生育组和不生育A、B、C组分别为 4、16、19和 5 ,生育组与不育组A、B组间的差异均有显著性 (P <0 .0 5或 0 .0 1)。结论　ACP活性大小与精子和膜的损伤密切相关 ,检测ACP活性可推断生精细胞和膜受损程度 ,提高诊断水平     

An enzyme-linked immunosorbent assay for the detection of hepatocyte plasma membrane antibodies

An enzyme-linked immunosorbent assay (ELISA) using plates coated with hepatocyte plasma membranes (HPM) was developed for the measurement of antibodies directed at hepatocyte surface antigens. Precoating ELISA plates with poly-L-lysine (PLL) provided firm attachment for the adsorption of HPM. The use of HPM, in preference to whole hepatocytes, excludes pathologically irrelevant cytoplasmic antigens. In addition, there is no necessity for glutaraldehyde fixation which is commonly used in cellular assays to maintain cellular integrity and which may result in loss or alteration in antigenic specificities. The assay was used to study loss of tolerance to mouse HPM in mice immunized with rat HPM. Three mouse strains were immunized, each strain developed antibodies to rat HPM and autoantibodies to mouse HPM with autoantibody levels reaching a peak 6-10 weeks after commencement of immunization. The correlation between ELISA and indirect immunofluorescence for the measurement of HPM autoantibodies was 0.79 (P less than 0.001) within the serum titration range of 1:25 to 1:200. Antibody to control kidney plasma membrane (KPM) was also measured by ELISA, after elimination of endogenous alkaline phosphatase activity using levamisole. Immunization with rat HPM elicited organ-non-specific autoantibodies to KPM, but these were at lower levels than autoantibodies to HPM.     

Postnatal development of semitendinosus muscle in the dog

In this study the differentiation of postnatal muscle fibres in dog semitendinosus muscle has been characterized. Several histochemical techniques for myosin ATPase and metabolic activity were used in animals aged between 1 day and 2 months. The results show that at birth there are two types of fibre, whose ATPase activity gradually changes during postnatal development, so that several types of fibre can be identified after 2 months. These finally become the four types of the adult: I, IIA, IIDog, IIC. The main conclusions of the study are that the use of mATPase techniques is appropriate for showing the differentiation between muscle fibres, even at early stages of postnatal development, and that the origin of the four different fibres of the adult can be traced back to early postnatal stages.     

Dystroglycan protein distribution coincides with basement membranes and muscle differentiation during mouse embryogenesis.

Using immunohistochemistry, we have examined beta-Dystroglycan protein distribution in the mouse embryo at embryonic stages E9.5 to E11.5. Our data show that Dystroglycan expression correlates with basement membranes in many tissues, such as the notochord, neural tube, promesonephros, and myotome. In the myotome, we describe the timing of Dystroglycan protein re-distribution at the surface of myogenic precursor cells as basement membrane assembles into a continuous sheet. We also report on non-basement-membrane-associated Dystroglycan expression in the floor plate and the myocardium. This distribution often corresponds to sites of expression previously reported in adults, suggesting that Dystroglycan is continuously produced during development.     

Morphology of the synovium during its differentiation and development in the mouse knee joint

Summary The prenatal and postnatal development of the mouse knee joint was investigated by transmission and scanning electron microscopy. In the prenatal stage, following the appearance of a narrow intercellular cleft between two skeletal elements on the 16th fetal day, clefting extended into the lateral synovial mesenchyme. In some regions, the extension of the cleft was very rapid, but in a certain region (future fat pad region), it was somewhat slower. Macrophage-like cells appeared in the synovial mesenchyme on the 16th fetal day, and then increased in number, and were distributed as if they were clustering around the presumptive clefting zone in the future fat pad region on the 17th–18th fetal day. This suggests that macrophage-like cells may participate in joint development, as they phagocytize and remove some kinds of solid extracellular matrix, and facilitate the cleft extension. In the early postnatal stage, scanning electron microscopic observations showed that there were two different types of cell in the synovial lining. One of them exhibited a surface morphology corresponding to that of macrophages: a spherical cell body and numerous pseudopodia. The other type of cell exhibited various cell shapes with many cytoplasmic processes extending along the synovial surface.     

F4/80-positive cells rapidly accumulate around tubuli recti and rete testis between 3 and 4 weeks of age in the mouse: an immunohistochemical study

PROBLEM: Previous studies demonstrated that F4/80 antigen (murine macrophage-specific antigen)-positive cells in testes of normal adult mice accumulate particularly in the interstitium adjacent to the tubuli recti and rete testis (i.e., the central region). However, it remains unknown whether this accumulation is a congenital or acquired phenomenon. METHOD OF STUDY: The distribution of F4/80-positive cells on frozen sections of testes obtained from various aged mice was immunohistochemically examined to determine when the positive cells specifically accumulate in the central region. RESULTS: F4/80-positive cells were homogeneously distributed throughout the testicular interstitium with no specific accumulation until 2 weeks of age. However, at 3 weeks of age, the density of positive cells in the central region became slightly, but significantly, higher than that in the interstitium between the seminiferous tubules. Between 3 and 4 weeks of age, the cell density in the central region increased rapidly, the density at 4 weeks of age reaching the level of the mature testes of 8-week-old mice. CONCLUSION: These results demonstrate that the specific accumulation of F4/80-positive cells in the central region is an acquired phenomenon, which starts and ends before puberty.     

Evidence for intussusceptive capillary growth in the chicken chorio-allantoic membrane (CAM)

Summary The aim of our investigations was to test whether the chicken chorio-allantoic membrane (CAM) could be an adequate in vivo model for a new mode of capillary growth, originally described in the rat lung and termed intussusceptive microvascular growth. According to that concept the capillary system does not grow by sprouting of vessels, but expands by insertion of transcapillary tissue pillars or posts which form new intercapillary meshes. In the present study, we observed slender transcapillary tissue pillars with diameters around 1 m in the CAM by in vivo microscopy, and analyzed their ultrastructure by transmission electron microscopic investigation of serial sections. The pillars corresponded in size to those previously described in rat lung microvasculature. On day 7, the pillar core contained endothelial-, endothelial-like cells and collagen fibers, and on day 12 additionally chorionic epithelial cells. As a hypothesis we propose that slender cytoplasmic extensions of endothelial cells, heavily interdigitated in the post area and often projecting into the vascular lumen, could initiate the first step of pillar formation, i.e., interconnect opposite capillary walls. During both stages of development endothelial-like cells were observed in close relationship with the pillars. These cells seem to be relevant for tissue post completion and growth, as they were found to invade the core of the pillars. From the localization of the interendothelial junctions in the post region, a certain similarity to the concept proposed for the lung can be found. The observations confirm that the CAM is a very suitable material for the in vivo investigation of intussusceptive capillary growth.A poster about this work presented at the meeting of the Union of the Swiss Societies for Experimental Biology was awarded the Gian Töndùry-Price 1990 of the Swiss Society for Anatomy, Histology and Embryology     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞线粒体膜电位的影响

为探讨双歧杆菌的完整肽聚糖(WPG)对小鼠腹腔巨噬细胞线粒体膜电位的影响,首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,用四甲基罗丹明乙酯(TMRE)标记线粒体膜电位,最后用激光共聚焦显微镜检测巨噬细胞内荧光强度的变化。和对照组相比,WPG刺激巨噬细胞后,巨噬细胞内反映线粒体膜电位的荧光强度明显增强。双歧杆菌的WPG在激活巨噬细胞的过程中可提高其线粒体膜电位。     

膜脂质构成对淋巴细胞膜特性及膜蛋白结构与功能的影响

本实验通过人为改变淋巴细胞胞膜脂质构成，进而研究了膜脂质，尤其是膜胆固醇，对淋巴细胞膜生物物理特性及膜蛋白结构，以及Ｃａ２＋通道蛋白活性的影响。结果显示，随着胞膜磷脂（ＰＬ）／胆固醇（Ｃｈ）摩尔比的降低，膜脂质的微粘滞性升高，活化淋巴细胞超极化反应增强，胞浆游离钙浓度［Ｃａ２＋］ｉ及Ｃａ２＋跨膜流动增加，且上述膜电位及Ｃａ２＋内流的变化可因Ｃａ２＋通道受阻而减弱或消失。提示膜脂质可通过调节Ｃａ２＋通道蛋白活性，进而影响Ｃａ２＋跨膜内流及膜电位。另外，膜脂质组成改变后，膜蛋白内源性荧光强度及最大发射波长峰位的变化，也表明膜脂质及相关膜特性可影响膜蛋白的结构与功能。     

The unique nucleotide sequence of the A beta gene in the NOD mouse is shared with its nondiabetic sister strains, the ILI and the CTS mouse

It is generally held that one of the recessive genes controlling diabetes in the NOD mouse is linked to the major histocompatibility complex (MHC). Unique substitution of Asp57 with Ser in the A beta chain is considered to make the A beta gene the MHC-linked susceptibility gene. We therefore analysed the nucleotide sequences of the A beta second exon in ILI, CTS, and NON mice, which are nondiabetic inbred strains but are derived from the same Jcl-ICR mice as the NOD mouse. The DNA sequence analyses revealed that the A beta second exon sequences in the ILI and CTS mice, but not in the NON mouse, are identical to that of the NOD mouse. Possible roles of Ser57 of the A beta chain in the nondiabetic sister strains are discussed.     

Experimental cerebral aneurysms in the female heterozygous Blotchy mouse

The Blotchy mouse is characterized by an X-linked inherited disorder of connective tissue synthesis. The susceptibility to aneurysm formation in the cerebral arteries of the circle of Willis was compared in female heterozygous 'Blotchy' and control mice subjected to unilateral carotid artery ligation either alone or associated with hypertension. Cerebral aneurysms developed only in hypertensive Blotchy mice (6/31 vs. 0/30 in hypertensive controls). Aneurysms of the aorta and its major branches occurred in normotensive mice only in the Blotchy group in which hypertension increased the incidence of mesenteric and coeliac aneurysms. A light microscopic study of interruptions of the internal elastic lamina (IIEL) showed that they developed in arteries of both Blotchy and control mice but to a greater extent in the Blotchy group where hypertension further increased their incidence. The IIEL incidence in the aortic arch varied in parallel to the occurrence of aneurysms in all the different arterial sites. Thus, in an apparently normally viable animal, the presence of a mutated gene which indirectly leads to defective elastin and collagen fibre synthesis, favours the formation of both peripheral and cerebral aneurysms. However, the development of cerebral aneurysms requires the addition of an increase in haemodynamic stress.