Lipid polarity and sorting in epithelial cells

Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier apparently resides in the outer, exoplasmic leaflet of the plasma membrane bilayer. First data are now available on the generation of these differences in Madin-Darby canine kidney (MDCK) cells, grown on filter supports. Experiments in which fluorescent precursors of apical lipids were introduced into the cell have demonstrated that upon biosynthesis apical lipids are sorted from basolateral lipids in an intracellular compartment. In this paper we present a model for the sorting process, the central point of which is that the two sets of lipids laterally segregate into microdomains that bud to form vesicles delivering the lipids to the apical and the basolateral plasma membrane domains, respectively.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.     

依赖于微环境的TGFβ信号

转化生长因子β（transforming growth factor β，TGFβ）家族成员是一类分泌的细胞因子，在胚胎发育、免疫调节、伤口修复、细胞增殖和抑制等过程中发挥重要作用。其中,TGFβ1、TGFβ2和TGFβ3在进化上最晚出现，并以潜伏态分泌。有别于家族中的大多数成员，TGFβ1-3的信号具有时空区域性，并依赖于其所处的微环境。阐明依赖于微环境的TGFβ信号的多层次调控机制对于理解TGFβ信号在免疫、癌症等生理、病理条件下的功能，以及开发临床治疗策略具有重要意义。本文从TGFβ前体复合物的结构入手，并从TGFβ的激活，配体-受体识别，跨膜信号传导及转录调控等方面，论述依赖于微环境的TGFβ信号的调控机制，同时讨论肿瘤微环境中多效的TGFβ信号，进而对今后的研究方向进行展望。     

Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor

A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF (1-34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1-31)NH2, (1-30)NH2 and (1-29)NH2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1-23)NH2, (1-22)NH2 and (1-21)NH2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1-19)NH2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3-21).     

Variants of the Varimax Rotation Method

In the present paper we present an example proving that the Varimax rotation method commonly used in factor analysis (see H. F. KAISER, 1958) need not give a unique solution. We also show that changing the original algorithm more than one local maximum of the criterion “V” can be achieved. Hence the loadings of factors and practical interpretations of solutions are affected in this way.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.