Immunofluorescent localization of collagen types I,III and IV,fibronectin, laminin,and basement membrane proteoglycan in developing mouse skin

Summary The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14–18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components — type IV collagen, laminin and heparan sulphate proteoglycan — were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components — types I and III collagen, and fibronectin — was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.     

Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells.     

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Genomic organisation of the spinocerebellar ataxia type 7 (SCA7) gene responsible for autosomal dominant cerebellar ataxia with retinal degeneration

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) is a progressive neurodegenerative disorder caused by a CAG expansion in the spinocerebellar ataxia 7 (SCA7) gene. Here, we describe the genomic organisation of the human SCA7 gene. The exon-intron boundaries were identified by sequencing plasmid subclones of a P1 artificial chromosome (PAC) clone containing the entire SCA7 gene. We found 13 exons, ranging in size from 69 to 979 bp, with all exon-intron boundaries following the GT-AG rule. The ATG initiation codon at position 554 of the cDNA occurs in exon 3 at position 12 and the coding region extends to the first five codons of exon 13, with the CAG repeat being located in exon 3 starting at codon 30. The intron sizes were determined by long-distance polymerase chain reaction with primers from neighbouring exons and by restriction mapping of the SCA7 PAC clone. The introns varied in size from 233 bp to about 40 kb, resulting in an overall size estimate for the SCA7 gene of 140 kb. Sequence analysis of intron 7 (491 bp) revealed a polymorphic GT/AC repeat, a useful intragenic marker for SCA7 in segregation studies.     

What can we learn from noncoding regions of similarity between genomes?

Background  

In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.