A Novel Family of Serine/Threonine Kinases Participating in Spermiogenesis

The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of ~65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.     

A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5′ end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3′ untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.     

Long-term persistence of vaccine-derived aluminum hydroxide is associated with chronic cognitive dysfunction

Macrophagic myofasciitis (MMF) is an emerging condition, characterized by specific muscle lesions assessing long-term persistence of aluminum hydroxide within macrophages at the site of previous immunization. Affected patients mainly complain of arthromyalgias, chronic fatigue, and cognitive difficulties. We designed a comprehensive battery of neuropsychological tests to prospectively delineate MMF-associated cognitive dysfunction (MACD). Compared to control patients with arthritis and chronic pain, MMF patients had pronounced and specific cognitive impairment. MACD mainly affected (i) both visual and verbal memory; (ii) executive functions, including attention, working memory, and planning; and (iii) left ear extinction at dichotic listening test. Cognitive deficits did not correlate with pain, fatigue, depression, or disease duration. Pathophysiological mechanisms underlying MACD remain to be determined. In conclusion, long-term persistence of vaccine-derived aluminum hydroxide within the body assessed by MMF is associated with cognitive dysfunction, not solely due to chronic pain, fatigue and depression.     

Pain Reactivity and Plasma β-Endorphin in Children and Adolescents with Autistic Disorder

Background

Reports of reduced pain sensitivity in autism have prompted opioid theories of autism and have practical care ramifications. Our objective was to examine behavioral and physiological pain responses, plasma β-endorphin levels and their relationship in a large group of individuals with autism.

Methodology/Principal Findings

The study was conducted on 73 children and adolescents with autism and 115 normal individuals matched for age, sex and pubertal stage. Behavioral pain reactivity of individuals with autism was assessed in three observational situations (parents at home, two caregivers at day-care, a nurse and child psychiatrist during blood drawing), and compared to controls during venepuncture. Plasma β-endorphin concentrations were measured by radioimmunoassay. A high proportion of individuals with autism displayed absent or reduced behavioral pain reactivity at home (68.6%), at day-care (34.2%) and during venepuncture (55.6%). Despite their high rate of absent behavioral pain reactivity during venepuncture (41.3 vs. 8.7% of controls, P<0.0001), individuals with autism displayed a significantly increased heart rate in response to venepuncture (P<0.05). Moreover, this response (Δ heart rate) was significantly greater than for controls (mean±SEM; 6.4±2.5 vs. 1.3±0.8 beats/min, P<0.05). Plasma β-endorphin levels were higher in the autistic group (P<0.001) and were positively associated with autism severity (P<0.001) and heart rate before or after venepuncture (P<0.05), but not with behavioral pain reactivity.

Conclusions/Significance

The greater heart rate response to venepuncture and the elevated plasma β-endorphin found in individuals with autism reflect enhanced physiological and biological stress responses that are dissociated from observable emotional and behavioral reactions. The results suggest strongly that prior reports of reduced pain sensitivity in autism are related to a different mode of pain expression rather than to an insensitivity or endogenous analgesia, and do not support opioid theories of autism. Clinical care practice and hypotheses regarding underlying mechanisms need to assume that children with autism are sensitive to pain.     

UHRF1 regulates CDH1 via promoter associated non-coding RNAs in prostate cancer cells

Non-coding RNAs (ncRNAs) transcribed from the promoter and the downstream region can affect the expression of the corresponding coding genes. It has been shown that sense-directed ncRNAs arising from the promoter region of the E-cadherin gene (CDH1) mediate its repression. Here, we show that an antisense-directed ncRNA (paRCDH1-AS) transcribed from the CDH1 promoter is necessary for its expression. paRCDH1-AS acts as a hooking scaffold by recruiting the epigenetic regulators, UHRF1, DNMT3A, SUV39H1 and SUZ12, involved in CDH1 repression. The binding of epigenetic regulators to paCRDH1-AS, indeed, prevents their localization to the chromatin on CDH1 promoter. Moreover, paRCDH1-AS silencing induces CDH1 repression and a switch of the epigenetic profile on the promoter towards a more closed chromatin. Using bioinformatic and experimental approaches we defined that the promoter of the paRCDH1-AS is shared with the E-cadherin gene, showing a bidirectional promoter activity. We found that UHRF1 controls both CDH1 and paRCDH1-AS by directly binding this bidirectional promoter region. Our study provides evidences, for the first time, that UHRF1 recruitment can be affected by promoter-associated non-coding RNAs, opening new perspective regarding the role of UHRF1 in these complex regulatory networks.     

Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPKalpha2 but not AMPKalpha1

Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPKalpha1/alpha2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKalpha2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKalpha2 at the site of LKB1 phosphorylation (Thr172) or phosphorylation of acetyl-CoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPKalpha2, significant basal activity of AMPKalpha1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPKalpha1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPKalpha2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPKalpha1 in cardiac muscle.     

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1 , NbRae1 , which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1 -silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro . Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.