Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

Cell survival and proliferation are modified by insulin-like growth factor 2 between days 9 and 10 of mouse gestation

The size of mammalian species involves the interaction of multiple genetic modifiers that control the timing and extent of growth mechanisms. Disruption of the paternal allele of the imprinted embryonic gene coding for insulin-like growth factor 2 (IGF2, Igf2(+m/-p)), results in viable mice that are 60% the weight of wild-type littermates. Differences in weight are first detected at embryonic day (E) 11, and the growth deficit is maintained throughout life. We report the mechanisms that account for this unusual phenotype. In order to quantify growth, we used novel methods to generate single cell suspensions of post-implantation mouse embryos. We were then able to quantify cell number, cell proliferation and cell death between E8.5 and E11.5 using flow cytometry. Determination of total embryo cell number also allowed us to time litters by a method other than by plugging. Wild-type and Igf2(+m/-p) embryos accumulated similar total cell numbers up to E9.25, but cell number began to diverge by around E9.5, with significant differences by E11 (75% of wild type). A relative increase in pyknotic nuclei, sub-GI cytometry counts and caspase activity, all indicative of cell death, occurred in Igf2(+m/-p) embryos at E9.25, reverting to wild-type levels by E9.75. This was followed at E9.75 by a significant reduction in the proportion of cells in S phase, quantified by S-phase cytometry counts and BrdU labelling. No significant differences in cell size were detected. We conclude that the majority of the cell number differences between wild-type and Igf2(+m/-p) mice can be accounted for by modification of cell survival and proliferation during the period (E9 to E10) of post-implantation development.     

Gill-derived glands in glandulocaudine fishes (teleostei: Characidae: Glandulocaudinae)

The Glandulocaudinae is a subfamily of neotropical characid fishes from Central and South America. A unifying feature of the subfamily is the caudal gland, found almost exclusively in males. The gland consists of tissue on the base of the caudal fin covered in part by hypertrophied scales. Scale movement as the caudal fin is flexed appears to facilitate the release of chemical compounds from the glandular tissue. We describe here a different structure, found in the gill cavity of mature males in 12 of 17 glandulocaudine genera examined. Termed a gill gland, it develops as a male secondary sex character and appears morphologically suited to release chemical signals. The gland forms by the growth of tissue over and around 4-13 anterior gill filaments on the first gill arch, forming chambers with ventral openings. Within the gland chambers, gill secondary lamellae usually shorten and may disappear. When secondary lamellae persist, simple columnar epithelial cells develop between them. In the absence of secondary lamellae, the gland chambers are lined with a simple cuboidal or columnar epithelium. Gland size and the degree of gill modification vary among species. Gill glands appear absent in five glandulocaudine genera, suggesting character reversals based on current phylogenetic hypotheses and systematic classification. Gill gland morphology suggests that this structure releases chemical compounds into the gill current. The presence of gill glands only in mature males suggests a function in reproduction and/or male aggression. Together with studies of the caudal gland, this research suggests that chemical signals may play important roles in glandulocaudine reproduction.     

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex,a sequence-selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.     

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.     

Photoreceptor death: Spatiotemporal patterns arising from one-hit death kinetics and a diffusible cell death factor

Retinitis pigmentosa (RP) is an inherited disease affecting approximately 1: 4000 individuals in North America. It is characterized clinically by the gradual apoptotic death of photoreceptor cells that occurs nonuniformly across the surface of the retina. Recently, it has been demonstrated that the time of death of many individual photoreceptors is random, a fact that must be reconciled with the spatiotemporal patterns of photoreceptor degeneration that are observed in patients with RP. One possible explanation is that a diffusible toxic factor is released by dying photoreceptors and induces adjacent cells to likewise undergo apoptosis. To determine if such a mechanism can result in patchy distributions of photoreceptor death, as frequently observed in RP patients, we studied cell attrition produced by a bistable biochemical switch in an idealized one-dimensional retina. We found that with a reasonable choice of parameter values, our model was able to produce patterns of cell death resembling those observed in RP. In the context of this model, patches on the order of histologically observable size could develop from a single release event, but their rates of formation were independent of the concentration of toxic factor released. Instead, factor concentration affected the overall rate of cell death, the number of degenerating patches, and their distribution across the retina.     

Reptin and pontin antagonistically regulate heart growth in zebrafish embryos

Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway.     

Identification and action of juvenile hormone III from sexually mature alate females of the red imported fire ant,Solenopsis invicta

Analysis of extracts of hemolymph obtained from sexually mature alate females of Solenopsis invicta from monogyne colonies resulted in identification of juvenile hormone III (JH III). The average amount of JH III was 0.32±0.04 pmol/μmolof hemolymph. Topical application of 0.038 pmol of JH III was sufficient to stimulate alates to shed their wings in the presence of the queen. The time in which alates were induced to dealate decreased linearly with increasing concentrations of JH III from 0.038 to 3.8 pmol. However, higher JH III concentrations deviated from linearity and did not reach dealation times comparable with those that occur after mating flights. Thus, it appears that the mechanism of dealation that occurs when female alates are out of the influence of their queen is different from the one associated with mating flights. Application of 0.42 μmol of precocene II inhibited dealation of alates in queenless colonies. However, this inhibition was reversed after applying 38 pmol JH III to precocene-treated alates. The sizes of corpora allata (CA) from sexuals treated with JH III did not differ from those of controls. However, the sizes of CA were reduced in alates treated with precocene II. The results indicated that JH was important to dealation.