[Effect of androgen replacement therapy on mixed dry eye in a rabbit model].

Objective: To explore the effect of androgen replacement therapy in a rabbit dry eye model characterized by androgen deficiency and meibomian gland dysfunction (MGD). Methods: Thirty 6-month-old male Chinchilla rabbits were randomly divided into the treatment group, model group and control group, with 10 rabbits in each group. In the treatment and model groups, 2/3 of the meibomian gland openings were closed by cauterization with electric coagulation pen, and bilateral testes were removed. One gram gel containing 1% testosterone was applied for 28 days on the skin of rabbits in the treatment group since day 28 after the surgery. The model group and control group received transdermal petrolatum instead. Tear secretion, tear breakup time (TBUT), corneal fluorescein staining, and serum free testosterone level were monitored throughout the study period. The globes and eyelids were collected for hematoxylin-eosin staining and periodic acid-Schiff staining. Conjunctival tissue was tested for the expression of miRNA-744-5p and interleukin-6. Meibum was collected for fatty acid analysis. Results: Animals presented with typical dry eye signs and androgen deficiency. After 28-day androgen replacement therapy, compared with the model group, the treatment group had a significantly higher tear secretion rate [(14.7±5.2) vs (10.3±3.6) mm, P=0.001], higher TBUT [(15.0±4.2) vs (10.2±3.6) s, P=0.003], lower fluorescein staining score [0 (0, 1) vs 2 (1, 4), P<0.001], and higher goblet cell density at conjunctival fornix (27.2±7.6 vs 10.7±4.8, P<0.001). Additionally, compared with the model group, the conjunctiva of the treatment group expressed a significantly lower level of miRNA-744-5p (1.67±0.24 vs 2.63±0.58, P<0.001) and interleukin-6 [2.38 (1.84, 4.61) vs 4.82 (3.99, 6.36), P=0.022]. Meanwhile, the treatment group showed significantly increased level of 16∶1, Δ9 fatty acid [(10.31±1.00)% vs (3.87±0.45)%, P<0.001] and iso-18∶0 fatty acid [(7.09±0.93)% vs (2.44±0.70)%, P<0.001], but decreased level of iso-26∶0 fatty acid [(5.72±1.07)% vs (8.02±0.65)%, P<0.001] in the meibum compared with the model group. Conclusion: Androgen replacement therapy can alleviate dry eye signs in rabbits presented with combined androgen deficiency and MGD.

Protective action of honokiol, administered orally, against oxidative stress in brain of mice challenged with NMDA.

Neuroprotective effect of honokiol (HK), orally administered, on oxidative damage in the brain of mice challenged with N-methyl-d-aspartic acid (NMDA) was examined. HK (1-100 mg/kg) was administered to Institute of Cancer Research (ICR) male mice through a gavage for 3 days consecutively, and on the third day, NMDA (150 mg/kg) was intraperitoneally (i.p.) administered. Administration of NMDA, causing a lethality of approximately 60%, resulted in a significant decrease of total glutathione (GSH) level and increase of thiobarbituric acid-reactive substances (TBARS) value in brain tissue. Meanwhile, oral administration of HK (> or = 3 mg/kg) for 3 days reduced the lethality (60%) in NMDA-treated group to 10% level, and alleviated the behavioral signs of NMDA neurotoxicity. Moreover, HK pretreatment restored the levels of total GSH and TBARS in the brain tissue to control levels (p<0.01). Additionally, GSH peroxidase activity in cytosolic portion of brain homogenate was also restored significantly (p<0.01), whereas GSH reductase activity was not. Separately, compared to vehicle-treated control, no significant changes in body and brain weight were observed in mice administered with HK. Based on these results, oral intake of HK is suggested to prevent oxidative stress in the brain of mice.

The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'.

Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 A resolution. This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point-group symmetry like ferritin and bacterioferritin from Escherichia coli. Crystallized in the I422 space group, its structural analysis reveals for the first time the non-symmetric heme molecule located on a twofold crystallographic symmetry axis. Other hemes of the protomer are situated on twofold noncrystallographic axes. Apparently, both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal. Five water molecules are situated close to the heme, which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues. Its ferroxidase center, in which Fe(II) is oxidized to Fe(III), is empty or fractionally occupied by a metal ion. Two positions are observed for the coordinating Glu18 side chain instead of one in the E. coli enzyme in which the site is occupied. This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.

[The genetic effect of estrogen receptor(ESR) on litter size traits in pig].

Litter size is one of the most important economic traits in pig production, and the more piglet numbers per litter is capable to increase pork production and bring more economic profit for pig industry. ESR (estrogen receptor) gene has been determined to be one of the major genes affecting phenotype of litter size without any genetic negative correlation to growth and carcass traits. An optimized standard PCR-RFLP protocol is employed to type 262 sows from 5 different breeds in ESR loci, and then with the computation based on linear model ESR gene is confirmed to be a major locus significantly associated with litter size (P < 0.001). The genetic effect of ESR gene is quite large in these breeds, especially in these Chinese pig population. The sows of beneficial homozygote BB produce 1.40-3.37 total number born/litter and 0.63-3.58 number born alive/litter more than the sows of non-beneficial homozygote AA do. The information found in the present study is very important and could be utilized as DNA marker for improvement of reproduction trait in practice of pig breeding.

Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains.

Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.

The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of ApoB-containing lipoproteins.

Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.

apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes.

apo B is a structural constituent of several classes of lipoprotein particles, including chylomicrons, VLDL, and LDL. To better understand the role of apo B in the body, we have used gene targeting in embryonic stem cells to create a null apo B allele in the mouse. Homozygous apo B deficiency led to embryonic lethality, with resorption of all embryos by gestational day 9. Heterozygotes showed an increased tendency to intrauterine death with some fetuses having incomplete neural tube closure and some live-born heterozygotes developing hydrocephalus. The majority of male heterozygotes were sterile, although the genitourinary system and sperm were grossly normal. Viable heterozygotes had normal triglycerides, but total, LDL, and HDL cholesterol levels were decreased by 37, 37, and 39%, respectively. Hepatic and intestinal apo B mRNA levels were decreased in heterozygotes, presumably contributing to the decreased LDL levels through decreased synthesis of apo B-containing lipoproteins. Kinetic studies indicated that heterozygotes had decreased transport rates of HDL cholesterol ester and apo A-I. As liver and intestinal apo A-I mRNA levels were unchanged, the mechanism for decreased apo A-I transport must be posttranscriptional. Heterozygotes also had normal cholesterol absorption and a normal response of the plasma lipoprotein pattern to chronic consumption of a high fat, high cholesterol, Western-type diet. In summary, we report a mouse model for apo B deficiency with several phenotypic features that were unexpected based on clinical studies of apo B-deficient humans, such as embryonic lethality in homozygotes and neural tube closure defects, male infertility, and a major defect in HDL production in heterozygotes. This model presents an opportunity to study the mechanisms underlying these phenotypic changes.

Apoprotein B100, an inefficiently translocated secretory protein, is bound to the cytosolic chaperone, heat shock protein 70.

Apoprotein B100 (apoB) is a secretory protein that appears to be constitutively translated but inefficiently translocated into the lumen of the endoplasmic reticulum. Using several experimental approaches, we found that apoB is bound to the cytosolic chaperone protein, heat shock protein 72/73 (commonly referred to as Hsp70). Similar to other chaperone-protein interactions, this binding was transient and ATP-sensitive. The binding of apoB to Hsp70 in HepG2 cells was decreased by treatment with oleic acid, which increases both translocation and secretion of apoB, and was increased by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor which efficiently protects apoB from cellular degradation without affecting translocation. The N-terminal 16% of apoB, which is efficiently translocated into the endoplasmic reticulum lumen in stably transfected Chinese hamster ovary (CHO) cells, showed minimal, if any, binding to Hsp70. The N-terminal 50% of apoB, which is very poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggest that domains of nascent apoB localized on the C-terminal regions of the molecule are transiently exposed to the cytosol during translation and/or translocation, and that Hsp70 functions as a molecular chaperone to maintain apoB in a translocational competent conformation until translocation is completed.

Alternative polyadenylation of apolipoprotein B RNA is a major cause of B-48 protein formation in rat hepatoma cell lines transfected with human apoB-100 minigenes.

The human apoB gene encodes an mRNA of 14121 nucleotides. In liver the apoB gene products a full-length mature protein of 4,536 amino acids (B-100), whereas in the intestine this gene produces a truncated protein of 2,152 amino acids (B-48). B-48 results from RNA editing of nucleotide 6666 from C to U, thereby producing a stop codon at position 2153. Rat liver has been shown to contain apoB RNA editing capability resulting in production of both B-100 and B-48. To create an in vitro expression system for human B-100, a minigene with a wild type coding sequence for the entire B-100 protein (B-100/Gln) was stably transfected into rat hepatoma cells (McA-RH7777). Similarly, a minigene with mutation at nucleotide 6667 that allowed translation even after editing of nucleotide 6666 (B-100/Leu, nonstop mutant), a minigene with an additional nonsense mutation at nucleotide 7053 to produce B-50 (B-50/Leu), and a truncated wild type minigene with a stop signal at codon 3261 to produce B-74 and an mRNA of 10 kb (B-74/Gln) were also transfected. Very little full-length B-100 and B-74 was produced by any of the respective constructions, including the B-100/Leu with the nonstop mutation. Transfection with B-100/Gln, B-100/Leu and B-74/Gln constructions produced greater than 90% of apoB as B-48, whereas the B-50/Leu construction produced 76% B-50 and 24% B-48. The inability of the B-100/Leu construction to produce B-100 suggested an explanation for B-48 production other than RNA editing. Northern blot analysis showed that the RNA produced by all four transfectants was shortened to a size of about 7 kb. A 10-kb but no 7-kb RNA was observed in the B-74/Leu construction when transfected to Chinese hamster ovary cells suggesting cell type specificity in generation of a shortened RNA. The 3' end of apoB RNA from McA-RH7777 B-100/Leu transfectants was reverse transcribed, cloned, and sequenced. This revealed two species of RNA: one polyadenylated at or near nucleotide 6775 capable of coding for B-48, the other polyadenylated at nucleotide 7080 capable of coding for B-50. In 18% of the cDNA clones, nucleotide 6666 was edited from C to T. In 6 of 34 clones, addition of the poly(A) tail after nucleotide 6774 created a TAA stop codon, whereas no stop signals could be detected in the remaining clones.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue.

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.