Signals causing change in morphological phenotype,growth mode,and gene expression of vascular endothelial cells

Comparison of three different lines of bovine aortal endothelial cells provides a clear demonstration of reversible morphologic phenotype coincidental with change in expression and growth mode. These phenotypic forms can be externally controlled so that cells may exist either in an epithelioid contact-inhibitable state or as a fibroblastoid non-contact-inhibitable state. Clonal cell line N (normal) shows a strong tendency to maintain the epithelioid phenotype. Clonal cell line Sp (sprout) can readily and reversibly adopt the epithelioid or fibroblastoid phenotype. A factor in normal serum is responsible for maintaining the cells in the epithelioid phenotype. This factor could be a growth factor since several polypeptide growth factors are shown to drive cells from the fibroblastoid phenotype to the epithelioid phenotype within 11 hours. This growth factor-induced change is not mediated through induced DNA synthesis. Clonal cell line V (variant) normally maintains the fibroblastoid phenotype but can be directed to the epithelioid phenotype provided cells are on an appropriate collagenous matrix. Associated with these changes in morphological phenotype are depression of the expression of the pro α₂ chain of collagen type I which may be characteristic of the contact-inhibited state and of an 80,000 mol wt polypeptide synthesized only by cells in the fibroblastoid phenotype. An endothelial cell collagen ECl (mol wt 177,000) was synthesized by all cell lines regardless of phenotype whereas a suspected breakdown product EC3 (mol wt 100,000) was found only in the epithelioid phenotype. Other differences and similarities between cell lines include expression of a 135,000 mol wt glycoprotein GP (V and N), the procollagen of collagen type III (N) of fibronectin (N, V, Sp), and of the pro α₁ chain of collagen type I (Sp, V). The characteristic expression of each line and its response to signals controlling morphologic phenotype impinges on the question of whether there exist several distinct types of vascular endothelial cells with different functional potentials controlled by extracellular signals.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express α-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.     

Morphological and physiological studies of rat kidney cortex slices undergoing isosmotic swelling and its reversal: A possible mechanism for ouabain-resistant control of cell volume

Summary Slices of rat kidney cortex were induced to swell by preincubation at 1°C in an isotonic Ringer's solution, and their capacity to reverse swelling, by net extrusion of cellular water, was studied during subsequent incubation at 25°C. The recovery from swelling was prevented by the respiratory inhibitor, antimycin A. On the other hand, extrusion of water was little affected by ouabain. The extrusion of water continuing in the presence of ouabain (but not that in its absence) was significantly reduced when furosemide was added or when medium Cl^– was replaced by NO ₃ ^– , or I^–. There was substantial variability in the morphological appearance of cells within the cortical slices. Different segments of the nephron showed different structural changes during swelling and its reversal, the proximal tubules being most markedly affected. Proximal tubular cells of swollen slices showed disorganization of brush borders and expansion of their apical surfaces, and contained vesicles in their apical cytoplasm. Upon recovery at 25°C, the apical portions of these cells showed reversal of the expansion, but some apical vesicles remained. These vesicles were much more numerous after recovery in the presence of ouabain, but they were much reduced in numbers, or totally absent, when recovery took place in the presence of furosemide or absence of Cl^–, with or without ouabain. The vesicles seen in the presence of ouabain alone appeared to fuse with each other and with infoldings of the basolateral plasma membrane. Rather similar results were obtained with distal tubular cells in the slices. We suggest that volume regulation in the proximal and distal tubular cells proceeds by way of two mechanisms. The first consists of extrusion of water coupled to the ouabain-sensitive transport of Na⁺ and K⁺. The other proceeds by way of an ouabain-resistant entry of water into apical cytoplasmic vesicles, following furosemide-sensitive movements of Cl^– and Na⁺; the vesicles then expel their contents by exocytosis at the basolateral cell borders.     

Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts

Summary Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n=6) and OI cell strains (n=10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300xmagnification). Our results show that: (i) OI fibroblasts take longer (11–27 days, mean 20 days) than control cells (10–19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87x10⁶ cells/P60, range 0.3–1.6x10⁶) at stationary phase relative to control cells (2.2x10⁶ cells/P60, range 1.7–2.6x10⁶; F_4,77=56.1, p<0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width: length ratio) was also abnormal in OI cells. (F_4,730=37.6, p<0.01), types I and II OI cells have significantly increased ratios (p<0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.Publication No. 84013 from the Montreal Children's Hospital Research Institute     

Tumour–stroma interactions in carcinogenesis: Basic aspects and perspectives

In contrast to the conventional notion regarding tumour development as a cell autonomous process in which the major participants were the cancer cells, increasing evidence attributes important role in the stromal components, namely fibroblasts, and view the tumour as a heterogenous mixture of different cell types. These different types of cells, being cancer cells, fibroblasts, endothelial cells, and others, interact reciprocally and play an almost equally important role in the manifestation of certain aspects of the malignant phenotype. The elucidation of the mechanistic base of such interactions, besides the contribution to understand fundamental aspects of tumour cell biology, promises important applications in diagnosis, prognosis and therapy of the disease. (Mol Cell Biochem 261: 117–122, 2004)     

Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation

Tissue inhibitor of metalloproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of ³H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 ± 12 nM (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture platic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo. © 1993 Wiley-Liss, Inc.     

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm² on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10^-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.     

新型复合血管制备中内皮细胞培养的初步研究

目的:为制备新型复合血管,对内皮细胞的体外培养进行研究。材料和方法:新生儿脐带静脉48条,分别应用酶消化法,贴壁法和机械刮取法获得内皮细胞,以含20％的胎牛血清M199液进行培养,相差镜下观察其贴壁和生长规律。结果:以酶灌注法优于内膜刮取法和内膜贴壁法;而酶灌注法以消化15至20分钟获取细胞和贴壁较多,采取两次消比法较一次消化法效果为佳。结论:三种方法获取和培养的内皮细胞,在数量上用于制备新型复合血管受到一定限制,有关研究有待进一步探讨。     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

Dose-dependent effect of radiation on angiogenic and angiostatic CXC chemokine expression in human endothelial cells

Blood vessel growth is regulated by angiogenic and angiostatic CXC chemokines, and radiation is a vasculogenic stimulus. We investigated the effect of radiation on endothelial cell chemokine signaling, receptor expression, and migration and apoptosis. Human umbilical vein endothelial cells were exposed to a single fraction of 0, 5, or 20 Gy of ionizing radiation (IR). All vasculogenic chemokines (CXCL1–3/5–8) increased 3–13-fold after 5 or 20 Gy IR. 20 Gy induced a marked increase (1.6–4-fold) in angiostatic CXC chemokines. CXCR4 expression increased 3.5 and 7-fold at 48 h after 5 and 20 Gy, respectively. Bone marrow progenitor cell chemotaxis was augmented by conditioned media from cells treated with 5 Gy IR. Whereas 5 Gy markedly decreased intrinsic cell apoptosis (0 Gy = 16% ± 3.6 vs. 5 Gy = 4.5% ± 0.3), 20 Gy increased it (21.4% ± 1.2); a reflection of pro-survival angiogenic chemokine expression. Radiation induces a dose-dependent increase in pro-angiogenic CXC chemokines and CXCR4. In contrast, angiostatic chemokines and apoptosis were induced at higher (20 Gy) radiation doses. Cell migration improved significantly following 5 Gy, but not 20 Gy IR. Collectively, these data suggest that lower doses of IR induce an angiogenic cascade while higher doses produce an angiostatic profile.     

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1 coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.     

Effects of angiotensin II on bone cells in vitro

Other than its known effects on the cardiovascular system, angiotensin II (Ang II) stimulates cell growth in several cell types. In this study, we examined whether it also might affect bone cell metabolism. Ang II stimulated DNA and collagen synthesis and decreased alkaline phosphatase (AP) activity in bone cell populations derived from the periosteum of fetal rat calvariae. Similar effects of Ang II were observed on human adult bone cells obtained by collagenase digestion from trabecular bone. Clonal cell analysis, autoradiographic studies, and receptor subtype analysis suggested the presence of specific Ang II receptor subtype 1 (AT₁) binding sites on AP⁺ osteoblastic precursor cells. Ang II had no direct effects on osteoblastic cells with a mature phenotype, but paracrine effects of Ang II on mature osteoblasts could be observed upon coculture with Ang II-responsive bone cell populations. Because Ang II is known to be locally generated by endothelial cells, Ang II might play an important role in coordinating capillary cell growth and osteoblastic bone formation during bone remodeling. J. Cell. Physiol. 175:89–98, 1998. © 1998 Wiley-Liss, Inc.     

Immuno-electron-microscopic study of the prolactin cells in the pituitary gland of male Wistar rats during aging

Summary Prolactin cells were identified by means of immunocytochemistry with protein-A gold as a marker on ultrathin sections of the pituitary gland of young (3–4 months), middle-aged (16–19 months), and aged (26–30 months) male Wistar rats. Point-counting volumetry revealed that the prolactin (PRL) cell-volume density in middle-aged rats was significantly increased in comparison to the volume densities in young and aged rats. Within the PRL-cell population, four types of PRL cells were distinguished on the basis of the shape and size of their secretory granules. During aging, dramatic changes occurred in the relative volumes of the four cell types. The volume percentage of cells with round granules (type I, granule diameter 150–250 nm, and type IIA, granule diameter 250–350 nm) increased from ±30% in young rats to ±90% in old rats. The volume percentage of cells with round and polymorphic granules (type IIB; granule diameter 350–400 nm and type III; granule diameter 500–600 nm) decreased from ±70% in young rats to ±7% in old rats. Age-related changes in serum PRL levels were not found. It is concluded that although during the life span of the male Wistar rat considerable changes in PRL-cell volume densities and in the ratios of PRL-cell types occur serum, PRL levels remain more or less constant.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue

Summary A method for the isolation and long-term culture of human microvessel endothelial cells from mammary adipose tissue (HuMMEC) obtained at breast reduction surgery has been developed. Pure cultures of HuMMEC were isolated by sequential digestion of the fat with collagenase and trypsin followed by specific selection of microvessel fragments withUlex europaeus agglutinin-1 coated magnetic beads (Dynabeads). The resulting cells formed contact-inhibited monolayers on gelatin and fibronectin substrates and capillary-like “tubes” on Matrigel; they also expressed von Willebrand factor, angiotensin-converting enzyme, and accumulated acetylated low density lipoprotein. Further immunofluorescence characterization revealed the presence of antigens for the endothelial cell specific monoclonal antibodies EN4 and H4-7/33. In addition, the origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (CD31), and endothelial leukocyte adhesion molecule-1 (ELAM-1/E-selectin) upon stimulation with tumor necrosis factor (TNF)α. HuMMEC were found to express-1 ELAM-1 at lower levels of TNFα (<10 ng/ml) than required by human umbilical vein endothelial cells. These cells should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.     

Studies on the leaf of Amaranthus retroflexus (Amaranthaceae): ultrastructure,plasmodesmatal frequency,and solute concentration in relation to phloem loading

Both the mesophyll and bundle-sheath cells associated with the minor veins in the leaf of Amaranthus retroflexus L. contain abundant tubular endoplasmic reticulum, which is continuous between the two cell types via numerous plasmodesmata in their common walls. In bundle-sheath cells, the tubular endoplasmic reticulum forms an extensive network that permeates the cytoplasm, and is closely associated, if not continuous, with the delimiting membranes of the chloroplasts, mitochondria, and microbodies. Both the number and frequency of plasmodesmata between various cell types decrease markedly from the bundle-sheath — vascular-parenchyma cell interface to the sicve-tube member — companion-cell interface. For plants taken directly from lighted growth chambers, a stronger mannitol solution (1.4 M) was required to plasmolyze the companion cells and sieve-tube members than that (0.6 M) necessary to plasmolyze the mesophyll, bundle-sheath, and vascular-parenchyma cells. Placing plants in the dark for 48 h reduced the solute concentration in all cell types. Judging from the frequency of plasmodesmata between the various cell types of the vascular bundles, and from the solute concentrations of the various cell types, it appears that assimilates are actively accumulated by the sieve-tube — companion-cell complex from the apoplast.     

Study on established lymphoid cells in maple syrup urine disease. Correlation with clinical heterogeneity

Summary Branched-chain keto acid dehydrogenase complex (BCKAD) was measured in lymphoid cells established from five patients with maple syrup urine disease (MSUD) and six control subjects. Two other MSUD lymphoid cell lines obtained from The Human Genetic Mutant Cell Repository were used as references. One day after subculture, the cells grew logarithmically up to 4–5 days. With this cell growth, BCKAD activity increased greatly in controls, but not in MSUD cells. The maximum BCKAD activity of MSUD cells was less than 7% and 13%–16% of the control in classic and variatant types, respectively. Leucine added to culture medium at the concentration of 10–20 mM significantly inhibited cell growth in MSUD cells alone, and with increasing concentration and impaired enzyme activity in a cell line, the effect became more prominent. The effects of isoleucine and valine were mild and did not differ between control and MSUD cells.This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (56480191; Matsuda)     

豚鼠肾小管上皮原代细胞的培养

目的建立一种简便易行的豚鼠原代肾小管上皮细胞培养方法。方法运用筛网分离法和多种酶消化法获取高纯度的肾小管上皮细胞。利用免疫组化法和形态学观察法鉴定培养的肾小管上皮细胞性质及纯度。结果通过肾小管节段贴壁,胶原酶消化组织节段和细胞等方法,有效地促进肾小管原代细胞增殖;胰酶节段消化法的细胞贴壁效果稍差,细胞传代状态不理想;胰酶消化法则细胞贴壁较少,细胞生长状态较差。结论培养豚鼠原代。肾小管上皮细胞是可行的。     

Ultimobranchial gland of the domestic fowl

Summary The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150–300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500–800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.