Inducers of DNA synthesis present during mitosis of mammalian cells lacking G1 and G2 phases.

The cell cycle analysis of Chinese hamster lung fibroblast V79-8 line by the premature chromosome condensation method has confirmed the absence of measurable G1 and G2 periods. Sendai virus-mediated fusion of mitotic V79-8 cells with G1 phase HeLa cells resulted in the induction of both DNA synthesis and premature chromosome condensation in the latter, indicating the presence of the inducers of DNA synthesis above the critical level not only throughout S phase, as it is in HeLa, but also during mitosis of V79-8 cells. No initiation of DNA synthesis was observed when G1 phase HeLa cells were fused with mitotic CHO cells. These results indicate that the presence or absence of a G1 period in the cell cycle depends on the levels of the inducers of DNA synthesis present in the cell during mitosis.     

Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K. In the present work, using HTC-IR cells, we have found that insulin stimulation promotes the relocalization of Sam68 from the nucleus to the cytoplasm, and we have further studied the role of Sam68 in insulin receptor signaling complexes, by co-precipitating experiments. Thus, Sam68 is co-precipitated with p85 PI3K, IRS-1 and IR. The association of Sam68 with these complexes is mediated by the SH2 domains of PI3K. Moreover, we have found that Sam68 is a p120GAP associated protein after Tyr-phosphorylation by the IR. This association is mediated by the SH2 domains of GAP (preferentially the C-terminal SH2). Thus, Sam68 is linking p120GAP to PI3K signaling pathway. In fact, PI3K activity was increased in both anti-Sam68 and anti-GAP immmunoprecipitates upon insulin stimulation. We propose that the recruitment of the docking protein Sam68 to the PI3K pathway may serve to allow the association of other signaling molecules, i.e. p120GAP. In this way, these signaling complexes may modulate other signaling cascades of IR, such as p21Ras pathway.     

Expression of mammalian sterile 20-like kinase 1 and 2 and Yes-associated protein 1 proteins in triple-negative breast cancer and the clinicopathological significance

Background and aim:Mammalian sterile 20-like kinase 1 and 2 (MST1/2) and Yes-associated protein 1 (YAP1) are the core molecules of the Hippo signaling pathway, which have been found to be unbalanced in the occurrence of tumors and promote the development of the lesions. The present study aimed to investigate the expression of MST1/2 and YAP1 proteins in triple-negative breast cancer (TNBC) and their clinicopathological significance.Methods:Immunohistochemistry was used to detect the expression level of protein in tissues. According to the percentage of positive cells and staining intensity, the expression intensity of MST1/2 and YAP1 proteins in the tissue samples was scored, and the correlation between MST1/2 and the clinicopathological features of TNBC were discussed.Results:The expression of MST1/2 and YAP1 was associated with histological grade, metastasis, lymph node metastasis stage, and tumor node metastasis stage. The overexpression of YAP1 predicted a poor prognosis in terms of overall survival and disease-free survival time. The MST1/2 expression was associated with improved overall survival and disease free survival of the patients.Conclusion:MST1/2 and YAP1 may be used as prognostic indicators to evaluate the recurrence of TNBC and might become one of the new targets for breast cancer treatment.     

Characterization and cell cycle regulation of the related human telomeric proteins Pin2 and TRF1 suggest a role in mitosis

Telomeres are essential for preserving chromosome integrity during the cell cycle and have been specifically implicated in mitotic progression, but little is known about the signaling molecule(s) involved. The human telomeric repeat binding factor protein (TRF1) is shown to be important in regulating telomere length. However, nothing is known about its function and regulation during the cell cycle. The sequence of PIN2, one of three human genes (PIN1-3) we previously cloned whose products interact with the Aspergillus NIMA cell cycle regulatory protein kinase, reveals that it encodes a protein that is identical in sequence to TRF1 apart from an internal deletion of 20 amino acids; Pin2 and TRF1 may be derived from the same gene, PIN2/TRF1. However, in the cell Pin2 was found to be the major expressed product and to form homo- and heterodimers with TRF1; both dimers were localized at telomeres. Pin2 directly bound the human telomeric repeat DNA in vitro, and was localized to all telomeres uniformly in telomerase-positive cells. In contrast, in several cell lines that contain barely detectable telomerase activity, Pin2 was highly concentrated at only a few telomeres. Interestingly, the protein level of Pin2 was highly regulated during the cell cycle, being strikingly increased in G2+M and decreased in G1 cells. Moreover, overexpression of Pin2 resulted in an accumulation of HeLa cells in G2+M. These results indicate that Pin2 is the major human telomeric protein and is highly regulated during the cell cycle, with a possible role in mitosis. The results also suggest that Pin2/TRF1 may connect mitotic control to the telomere regulatory machinery whose deregulation has been implicated in cancer and aging.     

An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension

The 8D9 antigen, a cell surface protein isolated from chicken brain that is related to the L1 class of cell adhesion molecules, is shown to contain an activity that promotes the attachment of neurons and the outgrowth of neurites from chicken tecta and mouse cerebellum. When purified 8D9 antigen is attached to a nitrocellulose-coated substrate, neurons rapidly attach and extend unfasciculated neurites. Little or no attachment of astroglia, oligodendroglia, and fibroblast-like cells to the 8D9 antigen is observed. We propose that a function of the 8D9 antigen is that of a neurite extension-promoting substrate in axon fascicles and in regeneration of peripheral nerves.     

Sam68 and ERKs regulate leptin-induced expression of OB-Rb mRNA in C2C12 myotubes

Acute leptin treatment significantly increases the mRNA of the long isoform of leptin receptor (OB-Rb) in C2C12 myotubes after as little as 30 min, without affecting that of the short isoform (OB-Ra). The Sam68 STAR protein has been implicated in leptin signal transduction as an adaptor molecule useful to recruit other signalling proteins. We found that leptin increased Sam68 tyrosine-phosphorylation so decreasing its poly(U)-binding capacity. RT-PCR analysis of the mRNA bound to immunoprecipitated Sam68 showed that Sam68 associated with OB-Rb but not OB-Ra mRNA in control and leptin-treated C2C12 cells. The siRNA-mediated silencing of Sam68 reduced its levels by 89% and abolished the leptin-mediated increase in OB-Rb mRNA. Leptin activates ERKs which in turn might phosphorylate Sam68 modifying its influence on mRNA. We did not observe any changes in Sam68 Ser/Thr phosphorylation but using the specific MEK1 inhibitor PD-98059 showed that leptin-mediated ERK activation is essential for leptin's effect on OB-Rb mRNA expression. Thus it appears that leptin has a positive short-term effect on the regulation of OB-Rb mRNA in C2C12 cells, involving both Sam68 and ERKs. These results might suggest that leptin signal acutely favours its own sensitivity.     

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.     

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis

Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.     

The Isolation and Characterization of a Broad Host Range Bcep22-like Podovirus JC1

Bacteriophage JC1 is a Podoviridae phage with a C1 morphotype, isolated on host strain Burkholderia cenocepacia Van1. Phage JC1 is capable of infecting an expansive range of Burkholderia cepacia complex (Bcc) species. The JC1 genome exhibits significant similarity and synteny to Bcep22-like phages and to many Ralstonia phages. The genome of JC1 was determined to be 61,182 bp in length with a 65.4% G + C content and is predicted to encode 76 proteins and 1 tRNA gene. Unlike the other Lessieviruses, JC1 encodes a putative helicase gene in its replication module, and it is in a unique organization not found in previously analyzed phages. The JC1 genome also harbours 3 interesting moron genes, that encode a carbon storage regulator (CsrA), an N-acetyltransferase, and a phosphoadenosine phosphosulfate (PAPS) reductase. JC1 can stably lysogenize its host Van1 and integrates into the 5′ end of the gene rimO. This is the first account of stable integration identified for Bcep22-like phages. JC1 has a higher global virulence index at 37 °C than at 30 °C (0.8 and 0.21, respectively); however, infection efficiency and lysogen stability are not affected by a change in temperature, and no observable temperature-sensitive switch between lytic and lysogenic lifestyle appears to exist. Although JC1 can stably lysogenize its host, it possesses some desirable characteristics for use in phage therapy. Phage JC1 has a broad host range and requires the inner core of the bacterial LPS for infection. Bacteria that mutate to evade infection by JC1 may develop a fitness disadvantage as seen in previously characterized LPS mutants lacking inner core.     

Cows' milk proteins cause similar Th1- and Th2-like immune response in diabetic and healthy children

Aims/hypothesis: Cows' milk proteins have been proposed to play a part in the pathogenesis of Type I (insulin-dependent) diabetes mellitus but both epidemiological and immunological studies have given conflicting results. Thus we aimed to study the immunological response to cows' milk proteins among diabetic and healthy children, focusing on the balance of Th1- and Th2-like lymphocytes. Methods: Peripheral blood mononuclear cells from 30 Type I diabetic children (4 to 18 years old) were examined and compared with peripheral blood mononuclear cells from 18 healthy age-matched control children (7 to 15 years old). Expression of IFN-γ and IL-4 mRNA were detected by realtime RT-PCR and as protein by ELISA after stimulation with BSA, the ABBOS-peptide (a. a. 152–169) and β-lactoglobulin (βLG) from cows' milk and ovalbumin from hens' egg. Phytohaemagglutinin and keyhole limpet haemocyanin were used as positive and negative controls, respectively. Results: Bovine serum albumin caused a weak Th2-like response in Type I diabetic children, whereas BSA antibodies decreased with age only among healthy children. Otherwise, cows' milk proteins (BSA, ABBOS and βLG) caused increased expression for IFN-γ and IL-4 mRNA in diabetic and healthy children. βLG caused the strongest immunological response, which decreased with age only among diabetic children. However, ovalbumin from egg caused a similar activation of the immune system and the immune response was similar in both diabetic and healthy children. Conclusion/interpretation: Proteins from cows' milk caused an equal Th1- and Th2-like immune response in diabetic and healthy children. Thus, our results do not support the hypothesis that cows' milk antigens are important for the immune process associated with Type I diabetes. [Diabetologia (2001) 44: 1140–1147] Received: 6 February 2001 and in revised form: 14 May 2001     

Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.     

Analysis of dynamin isoforms in mammalian brain: dynamin-1 expression is spatially and temporally regulated during postnatal development.

In adult rat brain, the microtubule-associated protein dynamin is composed of a closely spaced polypeptide doublet of approximately 100 kDa. Using an antibody preparation that is monospecific for dynamin-1 (the higher molecular mass isoform) we examined the temporal and regional expression of dynamin-1 in developing rat brain. Analysis of whole rat brain homogenates established that prior to postnatal day 9, dynamin-1 was present only at very low levels and thereafter its expression steadily increased with adult levels being attained by postnatal day 23. In individual regions of the brain, dynamin-1 levels were highest in cortex, amygdala, and striatum, significantly lower in olfactory bulb, cerebellum, and midbrain, and lowest in brainstem. During postnatal development, each of the regions exhibited approximately the same time course of protein expression except for a slight lag in expression in olfactory bulb. The spatial and temporal patterns of expression of dynamin-1 correlate with the establishment and/or maintenance of mature neuronal structure and function rather than dendritic or axonal outgrowth.     

High mobility group box 1 is a novel substrate of dipeptidyl peptidase-IV

Aims/hypothesis

High mobility group box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. Previous studies have shown that topical application of HMGB1 to skin wounds of mouse models of diabetes enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. Dipeptidyl peptidase IV (DPP-IV/CD26) is a protease whose activity is increased in diabetes and whose inhibition improves glucose tolerance. Since HMGB1 contains potential DPP-IV cleavage sites, we determined whether HMGB1 may be a substrate for DPP-IV and whether DPP-IV-mediated cleavage may alter the biological activity of HMGB1.

Methods

Reversed phase HPLC, mass spectrometry and western blot analyses were performed to analyse and identify HMGB1 peptides generated following DPP-IV digestion. HMGB1 angiogenic functions in the presence of DPP-IV were evaluated in vitro and in vivo. HMGB1 protein was detected in the serum of type 2 diabetic patients before and after treatment with DPP-IV inhibitors.

Results

DPP-IV cleaved HMGB1 at its N-terminal region and affected its angiogenic functions. Specifically, DPP-IV inhibited HMGB1-induced endothelial cell migration and capillary-like structure formation, as well as HMGB1-mediated vascular network formation in Matrigel implants in mice. We had previously found that HMGB1 promoted endothelial cell migration through activation of extracellular regulated kinase signalling pathway. Here we showed that such an effect was abolished in the presence of DPP-IV. Finally, the N-terminal truncated form of HMGB1 was detected in the serum of type 2 diabetic patients, in whom DPP-IV inhibitors enhanced the levels of full-length HMGB1.

Conclusions/interpretation

DPP-IV cleaves HMGB1 and, via this mechanism, inhibits HMGB1 angiogenic activity. Treatment with DPP-IV inhibitors may enhance HMGB1 activity in diabetic patients, thereby improving angiogenesis in this condition.     

Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana. Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis, activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N- and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis. The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.Immune responses in plants generally start by receptor-mediated detection of nonself molecules that are conserved among different classes of microbes, both beneficial and pathogenic (1). These molecules often have essential functions in microbial fitness (2) and are known as microbe-associated molecular patterns (MAMPs). Upon their perception by the plant, MAMPs trigger basal immune responses (3), e.g., ethylene biosynthesis, production of reactive oxygen species, release of antimicrobial compounds (4), and in certain cases programmed cell death (2). Collectively, these responses contribute to resistance against nonadapted pathogens [MAMP-triggered immunity (MTI)].MAMPs of plant-infecting microbes have been described for bacteria, fungi, and oomycetes. Three characterized bacterial MAMPs are flagellin (5), EF-Tu (6), and peptidoglycan (7). Flagellin is the main protein of the bacterial flagellum, which is used by eubacteria for movement. A highly conserved fragment of 22 aa, named flg22 (5), is sufficient to activate MTI in Arabidopsis and other plant species. Elongation factor thermo unstable (EF-Tu) is an abundant and conserved bacterial protein that plays a central role in the elongation phase of protein synthesis. An 18-aa domain of EF-Tu, named elf18, is recognized as a MAMP in Brassicaceae species, but not in other tested plant families (6). Peptidoglycan (PGN), the third characterized bacterial MAMP, is a major structural component of most bacterial cell walls. PGN, consisting of strands of alternating N-acetylglucosamine and N-acetylmuramic acid residues, triggers immunity in Arabidopsis (7). An important fungal MAMP is chitin, a structural component of all fungal cell walls. Plants are able to recognize chitin, and fragments of 4–10 N-acetylglucosamine residues are the most potent inducers of defense (8). Recently, a second fungal MAMP was identified, a secreted polygalacturonase of Botrytis cinerea that triggers immunity in Arabidopsis (9).Four oomycete-derived MAMPs have been identified to date (10): (i) heptaglucoside fragments, originating from branched β-glucans that are major cell wall polysaccharides, and that trigger defense responses in many Fabaceous plants (11); (ii) glycoprotein 42, a calcium-dependent transglutaminase that functions in irreversible protein cross-linking and is abundant in Phytophthora cell walls, and a 13-aa peptide fragment thereof that elicit MTI responses in parsley (12) and potato (13); (iii) elicitins, secreted proteins with sterol-binding activity (14), which provoke necrosis in Nicotiana plants through induction of cell death (15); and (iv) the Phytophthora cellulose-binding elicitor lectin, which is thought to cause perturbation of the cell wall cellulose status, thereby triggering necrosis and MTI in tobacco and Arabidopsis (16, 17). Other groups of cell death-inducing proteins may also qualify as MAMPs based on their widespread occurrence among different pathogens (2), e.g., the Crinklers and the cytotoxic necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) (10).Two major NLP types are found in bacteria, fungi, and oomycetes (18, 19) and are known to cause rapid necrosis and ethylene production in many dicot, but not in monocot plant species (18, 20). Type 2 NLPs differ from type 1 by an additional conserved second cysteine bridge and putative calcium-binding domain (19). In Arabidopsis, cytotoxic NLPs were found to activate immunity-related gene expression, which strongly overlapped with that induced by flg22 (21, 22). However, it was suggested that immune responses resulted from cytotoxicity. Moreover, necrosis was only induced upon treatment with the complete NLP protein (23). In vitro, cytotoxic NLPs cause rapid leakage of dicot membrane-derived vesicles, suggesting a direct cytolytic activity (24). The immunogenic effect of NLPs was therefore suggested to result from direct cellular damage (24), or release of damage-associated molecular patterns (3).Several plant-infecting oomycetes have large expansions of NLPs in their genomes (25–27), suggesting that these proteins play an important role in the pathogen’s lifestyle. A clear virulence function was observed for NLP_Pcc of the rot bacterium Pectobacterium carotovorum (27). Also, individual deletion of two NLP genes in the fungus Verticillium dahliae resulted in reduced virulence on different host plants (28). Five other NLP genes in this fungus encode noncytotoxic proteins (29), a phenomenon that is also observed in oomycetes. When tested by transient expression in tobacco, necrosis was only induced by 1 out of 3 tested NLPs of Phytophthora infestans (30), 8 out of 33 NLPs of Phytophthora sojae (31), whereas not a single 1 of 10 NLPs of Hyaloperonospora arabidopsidis tested caused necrosis (26). In contrast to cytotoxic NLPs that are mainly expressed during necrotrophic stages of infection, noncytotoxic NLPs appear to be expressed early during infection (26, 30), suggesting they serve an, as-yet-unknown, function during penetration or initial colonization of the host.In our search for the biological function of noncytotoxic NLPs of H. arabidopsidis, transgenic HaNLP-expressing Arabidopsis plants were generated that were severely stunted. In this paper, we show that Arabidopsis responds to noncytotoxic HaNLPs and small peptide fragments thereof that are highly conserved in type 1 NLPs. The peptides activate ethylene production and other typical MAMP-triggered defense responses, but not tissue necrosis, indicating they act as a MAMP. NLPs are not restricted to a single class of microbes but present in a broad range of mostly plant-associated microbes (bacteria, fungi, and oomycetes) belonging to three kingdoms of life, making this a MAMP with an unprecedented broad taxonomic occurrence.