C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions

Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.     

The simultaneous presence of IL-1B and TNFA two-positions risk haplotypes enhances the susceptibility for celiac disease

To assess the joint contribution of interleukin 1 beta (IL-1B) and tumor necrosis factor alpha (TNFalpha) to the genetic risk of developing celiac disease (CD), we analyzed four biallelic polymorphisms of TNFA and IL-1B genes in 228 patients and 244 healthy controls. The individual contribution of TNFA -308A and IL-1B -511C alleles was weak (OR 1.47 and 1.66, respectively) and was null for TNFA -238 A/G and IL-1B +3953 C/T single nucleotide polymorphisms (SNPs). Due to the potential linkage disequilibrium between TNFA, human leukocyte antigen (HLA) -DQA1 and HLA-DQB1 genes, only individuals carrying DQ2 antigen (DQ2-positive) were considered to perform haplotype analyses. Two-position risk haplotypes were first defined by the combined presence of -511C and +3953T alleles for IL-1B (OR 9.402) or -308A and -238A alleles for TNFA (OR 15.389). The TNFA/IL-1B combined haplotype-stratified association analysis showed that the simultaneous presence of TNFA risk and IL-1B non-risk haplotypes (OR 13.32) but not TNFA non-risk and IL-1B risk haplotypes (OR 0.71) is associated with CD. Interestingly, our data suggest that the coexistence of both risk haplotypes seems to work synergistically (OR 29.59), which enhances the risk of developing CD.     

Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe

Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained. The current results, together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I.     

Gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary

Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.     

Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.Subject terms: Cell death in the nervous system, Neurodegeneration, Oligodendrocyte     

Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP⁺), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.     

Calbindin‐D28k and calretinin in chicken inner retina during postnatal development and neuroplasticity by dim red light

Members of the family of calcium binding proteins (CBPs) are involved in the buffering of calcium (Ca2+) by regulating how Ca2+ can operate within synapses or more globally in the entire cytoplasm and they are present in a particular arrangement in all types of retinal neurons. Calbindin D28k and calretinin belong to the family of CBPs and they are mainly co‐expressed with other CBPs. Calbindin D28k is expressed in doubles cones, bipolar cells and in a subpopulation of amacrine and ganglion neurons. Calretinin is present in horizontal cells as well as in a subpopulation of amacrine and ganglion neurons. Both proteins fill the soma at the inner nuclear layer and the neuronal projections at the inner plexiform layer. Moreover, calbindin D28k and calretinin have been associated with neuronal plasticity in the central nervous system. During pre and early postnatal visual development, the visual system shows high responsiveness to environmental influences. In this work we observed modifications in the pattern of stratification of calbindin immunoreactive neurons, as well as in the total amount of calbindin through the early postnatal development. In order to test whether or not calbindin is involved in retinal plasticity we analyzed phosphorylated p38 MAPK expression, which showed a decrease in p‐p38 MAPK, concomitant to the observed decrease of calbindin D28k. Results showed in this study suggest that calbindin is a molecule related with neuroplasticity, and we suggest that calbindin D28k has significant roles in neuroplastic changes in the retina, when retinas are stimulated with different light conditions. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 530–542, 2013     

Health evaluation of wild gentoo penguins (Pygoscelis papua) in the Antarctic Peninsula

Historically wildlife conservation was based on habitat protection and exploitation control. Only recently have diseases been considered an important issue. However, pathogens are usually described during or after disease outbreaks, but to determine which pathogens may be emerging, surveys of wildlife health are critical in a given time. This study deals with the health status of gentoo penguins Pygoscelis papua in two localities at the Antarctica Peninsula and one at Ardley Island off the South Shetland Islands. Cloacal swaps, fresh fecal samples, ectoparasites, and blood smears were collected. We examined and dissected 14 penguin corpses found dead. Fecal samples were positive for Campylobacter, Escherichia coli and in the carcasses four endoparasitic species were found: Diphyllobothrium sp. and Parorchites zederi, Corynosoma shackletoni and Stegophorus adeliae. The tick Ixodes uriae occurred in five of the examined penguins, and the louse Austrogoniodes gressitti on six birds. From the colony grounds, we collected 1,184 I. uriae. We recorded antibiotic-resistant bacteria, such as E. coli, in ecosystems where gentoo penguins breed. Cloacal samples (300) were negative for Chlamydia, as well as for Salmonella, Campylobacter, E. coli, Newcastle and Influenza viruses.