Knocking down PFL can improve myocardial ischemia/reperfusion injury in rats by up-regulating heat shock protein-20.

The article "Knocking down PFL can improve myocardial ischemia/reperfusion injury in rats by up-regulating heat shock protein-20, by R.-L. Yin, H. You, Y.-M. Wu, F.-L. Ye, W.-X. Gu, J. Shen, published in Eur Rev Med Pharmacol Sci 2019; 23 (17): 7619-7627-DOI: 10.26355/eurrev_201909_18885-PMID: 31539154" has been withdrawn from the authors since "due to some inaccuracies (some data cannot be repeated by our further research)". The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/18885.

Knocking down PFL can improve myocardial ischemia/reperfusion injury in rats by up-regulating heat shock protein-20.

OBJECTIVE: To investigate the effect and mechanism of long non-coding ribonucleic acid (lncRNA) PFL on myocardial ischemia/reperfusion (I/R) injury in rats, and to provide a reference for the prevention and treatment of myocardial infarction (MI) in clinic. MATERIALS AND METHODS: According to the random number table, 60 male Sprague-Dawley (SD) rats were randomly divided into 3 groups: Control group (n=20), I/R group (n=20), and I/R + PFL small interfering ribonucleic acid (siRNA) group (n=20). The I/R model was established by ligating the left anterior descending coronary artery (LAD) and then recanalizing it. PFL siRNAs were injected intravenously into the tail vein of rats in I/R + PFL siRNA group to construct a PFL knockout model. Triphenyl tetrazolium chloride (TTC) test was used to detect the infarction area of each group. Echocardiography was adopted to measure the ejection fraction [EF (%)] and fraction shortening [FS (%)] of rats in each group. Hematoxylin and eosin (H&E) staining was applied to detect the morphological changes in myocardial cells in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to detect the apoptosis levels of myocardial cells and fibroblasts in heart tissues in each group. Meanwhile, the protein expression levels of apoptosis-related genes, Bcl-2-associated X protein (BAX), and Bcl-2, were measured via Western blotting. Also, the expression level of heat shock protein 20 (HSP-20) in the heart of three groups of rats was examined using immunohistochemical staining. Finally, the effects of PFL siRNAs on the expression level of HSP-20 were detected via Western blotting. RESULTS: PFL siRNAs could significantly improve I/R-induced cardiac insufficiency in rats, thus increasing EF (%) and FS (%) (p<0.05). Besides, PFL siRNAs could remarkably inhibit cardiac infarction caused by I/R injury and reduce the infarction area from (59.54±3.45)% to (24.85±1.30)% (p<0.05). H&E staining results manifested that, compared with those in I/R group, the cardiac myofilament was better in alignment, degradation and necrosis were milder, and cell edema was notably reduced in I/R + PFL siRNA group. Immunohistochemistry and Western blotting results showed that PFL siRNAs could remarkably reverse the decrease in the HSP-20 expression caused by I/R (p<0.05). CONCLUSIONS: We found that PFL knockdown can significantly improve the myocardial injury caused by I/R and improve the cardiac function in rats. The mechanism may be related to the activation of HSP-20 by PFL siRNAs. Therefore, PFL is expected to become a new target for the treatment of MI.

Evidence for inbreeding depression in the tree Robinia pseudoacacia L. (Fabaceae).

The magnitude of inbreeding depression within populations is important for the evolution and maintenance of mixed mating systems. However, data are sparse on the magnitude of inbreeding depression in Robinia pseudoacacia. In this study, we compared differences in the mature seed set per fruit, seed mass, germination success, and seedling growth between self- and cross-pollination treatments and estimated the inbreeding depression at 3 stages: seed maturation, seedling emergence, and seedling growth at 10 and 20 weeks. We found that progenies resulting from cross-pollination treatments showed significantly higher fitness than progenies resulting from self-pollination, causing high levels of inbreeding depression. Inbreeding depression was not uniformly manifested, however, over the 3 stages. Inbreeding depression was the greatest between fertilization and seed maturation stage (δ = 0.5419), and the seedling emergence (0.3654) stage was second. No significant differences in seedling growth were observed between selfed and crossed progenies. The cumulative inbreeding depression (δ) across all 3 stages averaged 0.7452. Inbreeding depression may promote outcrossing in R. pseudoacacia by acting as a post-pollination barrier to selfing. The large difference in the seed set between self- and cross-pollination that we detected indicated that inbreeding depression would probably be a reasonable explanation for the high abortion and low seed set in R. pseudoacacia.

Mutual up-regulation of thyroid hormone and parathyroid hormone receptors in rat osteoblastic osteosarcoma 17/2.8 cells.

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.

A novel mutation (M310L) in the thyroid hormone receptor beta causing resistance to thyroid hormone in a Brazilian kindred and a neonate.

Resistance to thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to thyroid hormone (T3) caused by mutations in the thyroid hormone receptor beta (TRbeta). The index patient of the family reported here, a 17-year-old woman, came to medical attention because of a diffuse goiter, short stature, and learning disabilities. Biochemical tests revealed an elevated free T4 of 5.2 ng/dl (0.8-2.1), a T3 of 270 ng/dl (80-220), and a nonsuppressed TSH of 1.79 mU/l (0.4-4). Administration of exogenous T4 or T3 did not result in the usual TSH suppression, prompting the clinical diagnosis of RTH. Her father and one of her brothers also had clinical and biochemical findings consistent with RTH. Direct sequence analysis of the TRbeta gene revealed a heterozygous transition 928A>G in exon 9 resulting in substitution of methionine 310 by leucine (M310L). This novel receptor mutant has a reduced affinity for T3 ( approximately 10% of normal) and dominant negative properties that are similar in comparison to other RTH mutations. The index patient had a normal pregnancy and delivery. At birth, the female neonate had no goiter, a significantly elevated T4, and increased TSH. The diagnosis of RTH was confirmed by sequencing the TRbeta gene. She was underweight at birth and her length was between the 5th and 10th percentile. At 26 months, her height remained at the 10th percentile but her bone age was 18 months, suggesting mild hypothyroidism at the level of the bone. In contrast, increased heart rate and restlessness are consistent with hyperthyroidism in other tissues, such as the heart and possibly the brain.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

X-linked Kallmann syndrome and renal agenesis occurring together and independently in a large Australian family.

Males with X-linked Kallmann syndrome (XLKS) may have renal agenesis. We studied a large kindred with a history of eight males affected by XLKS born in five generations. Their XLKS was shown to be due to an intragenic mutation of the KAL-1 gene. We also documented three male neonatal deaths due to bilateral renal agenesis (BRA), five males with unilateral renal agenesis (URA), and one female with a pelvic ectopic kidney in this kindred. Of four XLKS males who had renal imaging studies, two had URA. The kindred's KAL-1 mutation was not present in three of the males with URA, the female with the ectopic kidney, nor in preserved autopsy tissue from one infant with BRA. The high frequency of renal agenesis in this family, in the presence and absence of the KAL-1 mutation, suggests an autosomal dominant or X-linked gene which may independently or co-dependently contribute to renal agenesis.

Clinical and functional effects of mutations in the DAX-1 gene in patients with adrenal hypoplasia congenita.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder caused by mutations in a gene referred to as DAX-1. AHC is characterized by adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. The DAX-1 protein is structurally related to orphan nuclear receptors, although it lacks the characteristic zinc finger DNA-binding domain that is highly conserved in other members of this family. In this report, we describe the clinical features and genetic alterations in six families with AHC. These patients reveal the variable clinical presentation of adrenal insufficiency in AHC and underscore the importance of considering this diagnosis. Nonsense mutations that introduce a stop codon were found in three cases (W171X, W171X, Y399X). Frameshift mutations (405delT, 501delA, and 702delC), each of which resulted in a premature stop codon at amino acid 263, were found in the other three families. Three of these mutations (Y399X, 405delT, 702delC) are novel. Using transient gene expression assays to assess DAX-1 function, these mutations were shown to eliminate the ability of DAX-1 to repress the transcription of genes that are stimulated by a related nuclear receptor, steroidogenic factor-1. These studies reveal the variable clinical presentation of DAX-1 mutations and emphasize the value of genetic testing in boys with primary adrenal insufficiency and suspected X-linked AHC.

A novel natural mutation in the thyroid hormone receptor defines a dual functional domain that exchanges nuclear receptor corepressors and coactivators.

In a patient with severe resistance to thyroid hormone (RTH), we found a novel mutation (leucine to serine in codon 454, L454S) of the thyroid hormone receptor beta. This mutation is in the ligand-dependent transactivation domain that has been shown to interact with transcriptional coactivators (CoAs). The mutant protein binds T3, but its ability to activate transcription of a positively regulated gene (TRE-tk-Luc), and to repress a negatively regulated gene (TSHalpha-Luc), is markedly impaired. As anticipated from its location, the L454S mutant interacts weakly with CoAs, such as SRC1 and glucocorticoid receptor interacting protein 1 (GRIP1) in gel mobility shift assays and in mammalian two-hybrid assays, even in the presence of the maximal dose of T3. In contrast, in the absence of T3, the L454S mutant interacts much more strongly with nuclear receptor corepressor (NCoR) than does the wild-type receptor, and the T3-dependent release of NCoR is markedly impaired. By comparison, the NCoR interaction and T3-dependent dissociation of an adjacent AF-2 domain mutant (E457A) are normal. These findings reveal that the Leu 454 is involved directly, or indirectly, in the release of corepressors (CoRs) as well as in the recruitment of CoAs. The strong interaction with NCoR at a physiological concentration of T3 results in constitutive activation of the TSH genes as well as constitutive silencing of positively regulated genes. When the dominant negative effect was examined among various mutants, it correlated surprisingly well with the potency of NCoR binding but not with the degree of impairment in CoA binding. These findings suggest that the defective release of NCoRs, along with retained dimerization and DNA binding, are critical features for the inhibitory action of mutant thyroid hormone receptors. These studies also suggest that helix 12 of the thyroid hormone receptor acts as a dual functional domain. After the binding of T3, its conformation changes, causing the disruption of CoR binding and the recruitment of CoAs.

A novel aminoterminal mutation in the KAL-1 gene in a large pedigree with X-linked Kallmann syndrome.

Kallmann syndrome is characterized by hypogonadotropic hypogonadism and anosmia. Autosomal dominant, autosomal recessive, and X-linked patterns of transmission have been described. The X-linked form of Kallmann syndrome (XLKS) is the least common of the three modes of inheritance and is caused by mutations in the putative cell adhesion protein, KAL-1. In a large pedigree with XLKS, direct sequencing of the KAL-1 gene revealed a duplication of 11 base pairs in exon 1, resulting in a frameshift and a premature stop at codon 34 of the 680 amino acid protein. The clinical features of the affected individuals in this pedigree provide further evidence in support of the idea that XLKS is associated with neurologic features that are not seen in other forms of the syndrome.

Congenital hyperthyroidism caused by a solitary toxic adenoma harboring a novel somatic mutation (serine281-->isoleucine) in the extracellular domain of the thyrotropin receptor.

Activating somatic mutations in the thyrotropin (TSH) receptor have been identified as a cause of hyperfunctioning thyroid adenomas, and germline mutations have been found in familial nonautoimmune hyperthyroidism and sporadic congenital hyperthyroidism. All mutations reported to date have been located in the transmembrane domain. We now report an example of an activating mutation in the extracellular, TSH-binding domain, found in a male infant with congenital hyperthyroidism due to a toxic adenoma. The pregnancy was remarkable for fetal tachycardia. Scintigraphic studies demonstrated a large nodule in the right lobe, and a hemithyroidectomy was performed at the age of 2 yr. Direct sequencing of the TSH receptor gene revealed a mutation in one allele resulting in a substitution of serine281 by isoleucine (Ser281--> Ile) in the extracellular domain. The mutation was restricted to the adenomatous tissue. Expression of the Ser281--> Ile mutation in vitro revealed an increase in basal cAMP levels. Affinity for TSH was increased by the mutation. These findings demonstrate that activating mutations can also occur in the extracellular domain of the TSH receptor, and support a model in which the extracellular domain serves to restrain receptor function in the absence of TSH or antibody-induced conformational changes.

The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding.

The hyt/hyt mouse is hypothyroid because of a mutation in the TSH receptor (TSH-R). In this report, we confirm the presence of a Pro to Leu mutation in amino acid 556 of the fourth transmembrane domain (TM4) of the TSH-R. This Pro is highly conserved in members of the G protein-coupled seven-transmembrane family of receptors. Insertion of this mutation into the wild-type rat receptor eliminated TSH binding and receptor function in transfected 293 and COS cells. Wild-type TSH-R conferred a 7.4-fold increase in cAMP and a 2.3-fold stimulation of a cAMP-responsive reporter gene. The P556L mutant receptor elicited no increase in cAMP or the reporter gene. Cells transfected with wild-type receptor bound TSH with a Kd of 3.3 x 10(-10) M, whereas no TSH binding was detected with the P556L mutant. Because the P556L mutation occurs in a receptor region (TM4) that is not expected to alter the binding of TSH, additional studies were performed to examine receptor processing and cellular localization. Mutant receptors from solubilized membranes also failed to bind TSH, indicating that the absence of binding to intact cells was not accounted for intracellular trapping of the mutant receptor. Western blot analyses demonstrated that the mutant and wild-type receptors were processed through a similar series of precursors and that a mature 95-kilodalton form of the mutant TSH-R was produced, consistent with its insertion into the plasma membrane. Immunofluorescence studies confirmed expression of the P556L mutant on the cell surface of transfected cells and in thyroid tissue from hyt/hyt mice. Although the extracellular domain of the TSH-R is sufficient for high affinity binding of TSH, we conclude that the hyt mutation in the fourth transmembrane domain eliminates TSH binding. These results suggest interactions between the extracellular and transmembrane domains of the TSH-R and indicate that this highly conserved proline is required for normal receptor structure and function.

[Effect of nisoldipine on cultured myocardial cells of neonatal rats].

Nisoldipine 1.5 nmol/L, 0.75, and 1.5 mumol/L depressed the beat rates of myocardial cells. The effects were negatively correlated with dosages. Nisoldipine depressed the beat rates increased by calcium chloride 0.15 mmol/L or isoprenaline 1.5 mumol/L and atropine 20 nmol/L. Conversely, the above drugs reversed the beat rates depressed by nisoldipine. Therefore, the acting nisoldipine may be antagonistic to calcium chloride, isoprenaline and atropine. In addition, nisoldipine 10 nmol/L may significantly aggravated the damage to myocardial cells induced by mitomycin C 5 micrograms/ml in which elevated lactate dehydrogenase appeared.

The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells.

[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides.