Changes at the nuclear lamina alter binding of pioneer factor Foxa2 in aged liver

Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age‐dependent metabolic changes, but the mechanism is unknown. Here, we profile lamina‐associated domains (LADs) using lamin B1 ChIP‐Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1‐associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24‐deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de‐repression of PPAR‐ and LXR‐dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de‐repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.     

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Composition in situ and in vitro of vascular smooth muscle laminin in the rat

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.     

Pro-fibrotic pathway activation in trabecular meshwork and lamina cribrosa is the main driving force of glaucoma

While primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, it still does not have a clear mechanism that can explain all clinical cases of the disease. Elevated IOP is associated with increased accumulation of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) that prevents normal outflow of aqueous humor (AH) and has damaging effects on the fine mesh-like lamina cribrosa (LC) through which the optic nerve fibers pass. Applying a pathway analysis algorithm, we discovered that an elevated level of TGFβ observed in glaucoma-affected tissues could lead to pro-fibrotic pathway activation in TM and in LC. In turn, activated pro-fibrotic pathways lead to ECM remodeling in TM and LC, making TM less efficient in AH drainage and making LC more susceptible to damage from elevated IOP via ECM transformation in LC. We propose pathway targets for potential therapeutic interventions to delay or avoid fibrosis initiation in TM and LC tissues.     

Cell degeneration and elimination in the imaginal wing disc,caused by the mutationsvestigial andUltravestigial ofDrosophila melanogaster

Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg ^U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg ^U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (apoptosis). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg ^U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

The Fine Structure of Four “Species” of Amoeba*

The fine structure of Amoeba discoides, Amoeba dubia, and Amoeba amazonas was studied and compared with that of Amoeba proteus. The different kinds of amebas showed general similarities but differed in the ultrastructural details of their organelles. With respect to fine structure, A. discoides was indistinguishable from A. proteus, while both A. dubia and A. amazonas had distinctive features. The nuclei of all had a prominent honeycomb-like fibrous lamina, but A. dubia differed from the others in the distribution of nucleoli within the nucleus. The mitochondria of A. amazonas were unusual in having a variable pattern of cristae, some being plate-like and others tubular. Golgi bodies in A. amazonas had a greater proportion of vesicles and a smaller number of cisternae than those of the others, while Golgi bodies in A. dubia had highly flattened cisternae without a lining of filamentous material such as is found in the other types. The plasma membrane of A. dubia also lacked the prominent filamentous cell coat common to A. proteus and other amebas. The relation between the Golgi apparatus and the cell coat and the significance of the degree of development of the cell coat for pinocytosis and other phenomena is considered. The experimental use of these cells, including the formation of hybrids by nuclear transplantation is discussed.     

Chromatographic and biologic analysis of ME and OVLT LHRH

The luteinizing hormone (LH)-releasing activities a pooled rat organum vasculosum lamina terminalis (OVLT) and median eminence (ME) tissues were evaluated for chromatographic and biologic similarity and compared to those of synthetic decapeptide LH-releasing hormone (LHRH). The LHRH detected in these extracts appeared similar chromatographically (Sephadex G-25) to synthetic LHRH. These extracts, as well as synthetic LHRH, were also capable of stimulating dose dependent gonadotropin release form cultures rat gonadotrophs. These findings suggest a physiological role of the LHRH present in the rat OVLT in the control of gonadotropin secretion.     

Cultural behavior of protoplasts from different organs of eggplant (solanum melongena L.), and plant regeneration

Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 Em^-2s^-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl^-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl^-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×10³ protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×10³ and 1,220.4×10³ protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl^-1) + zeatin (0.5 mgl^-1) + NAA (1 mgl^-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl^-1) + IAA (0.2 mgl^-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl^-1) + NAA (0.5 mgl^-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.Abbreviations (IAA) Indol-3-acetic acid - (2,4-D) 2,4-dichlorophenoxyacetic acid - (NAA) naphthale-neacetic - (BAP) 6-benzylaminopurine - (MS) Murashige and Skoog basal medium - (CPW) Cell and Protoplast Washing solution