CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3–7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate β-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with β-carotene reduced the amount of cross-linking. We conclude that the high levels of β-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Ultrastructural and immunofluorescence studies of basal-lamina alterations during mouse-lung morphogenesis

Epithelial differentiation during lung development appears to be influenced by mesenchyme-derived instructions coupled with hormonal regulations. The basal lamina which is associated with progenitor and differentiating epithelia during mouse embryogenesis (Theiler-stages 16-28) was examined by transmission electron microscopy and indirect-immunofluorescence microscopy. During the embryonic phase of lung development, progenitor epithelia for the pulmonary acinus projected microvilli or cytoplasmic "feet" through the basal lamina, which resulted in discontinuities and a close approximation of the adjacent mesenchymal-cell processes. These changes were also associated with the transitory polarization of mesenchymal cells perpendicular to the plane of the basal lamina, which resulted in a sheet of cuboidal mesenchymal cells adjacent to the developing acinar-tubule epithelium. During the embryonic phase of lung development, these specific interstitial or mesenchymal cells stained for heparan-sulfate proteoglycans; no other cell types were immunostained. By Theiler-stage 25, the acinar-tubule epithelia had differentiated into type-II pneumonocytes which contained lamellar bodies and significant amounts of glycogen. Fibronectin, laminin, and heparan-sulfate proteoglycan were localized in the basement membranes during the embryonic, canalicular, and terminal sac phases of lung morphogenesis. A diffuse localization of fibronectin of the interstitial cell surfaces was observed. These observations indicate that major changes in the structure and composition of basal lamina occur during the embryonic and fetal phases of pulmonary-acinus epithelial-cell differentiation and the production of pulmonary surfactant. The major changes in the basal lamina may be partly mediated by mesenchyme-derived instructions for type-II epithelial-cell differentiation.     

Corticosteroid-induced cleft palate in mice and H-2 haplotype: Maternal and embryonic effects

This report confirms and expands on the original preliminary observations made by Bonner and Slavkin that corticosteroid-induced cleft palate in mice is associated with H-2 haplotype. Using three congenic strains, B10, B10.A, and B10.D2, our studies demonstrate that B10.A (H-2 ^b) is most susceptible and B10.D2 (H-2 ^d) is least susceptible, B10 (H-2 ^b) being intermediate. Variation in fetal loss among strains accounts for less than 1 percent of the variation in cleft-palate frequency among strains; variation in H-2 haplotype, however, accounts for more than 60 percent of the variation in cleft-palate frequency. With regard to all possible reciprocal F₁ hybrids, our results indicate that while there is a significant maternal effect, maternal haplotype can account for only 11 percent of the variation in cleft-palate frequency among crosses. Embryonic haplotype accounts for 17 percent of the variation, which is indicative of an important embryonic effect. Finally, our studies suggest that susceptibility to corticosteroid-induced cleft palate is associated with the K end of the H-2 complex.     

Constitutive production of lymphocyte activating factors by normal tissues in the adult rat

Lymphocyte activating factors (LAFs), e.g., interleukin-1 (IL-1) and IL-1-like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL-1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoeitic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL-1 alpha and IL-1 beta. Dose response curves and gel filtration on a Sephacryl S-200 column suggested the presence of a high molecular weight (90,000-100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000-25,000 Da, as determined by Sephacryl S-200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

Chromatin modifiers and recombination factors promote a telomere fold-back structure,that is lost during replicative senescence

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.