Nernst Sign Reversal in the Hexatic Vortex Phase of Weakly Disordered a-MoGe Thin Films.

The hexatic phase is an intermediate stage in the melting process of a 2D crystal due to topological defects. Recently, this exotic phase was experimentally identified in the vortex lattice of 2D weakly disordered superconducting MoGe by scanning tunneling microscopic measurements. Here, we study this vortex state by the Nernst effect, which is an effective and sensitive tool to detect vortex motion, especially in the superconducting fluctuation regime. We find a surprising Nernst sign reversal at the melting transition of the hexatic phase. We propose that they are a consequence of vortex dislocations in the hexatic state which diffuse preferably from the cold to hot.

Prospective study of antenatally diagnosed congenital cystic adenomatoid malformations.

PURPOSE: Congenital cystic adenomatoid malformations (CCAMs) are increasingly diagnosed in recent years due to the routine usage of antenatal ultrasound. The aim of this study was to present the natural course and outcome of antenatally diagnosed cystic lung diseases, which were prospectively studied. METHODS: Between the period June 2004 and June 2010, 25 fetuses with suspected CCAMs were seen in a single tertiary maternal fetal unit. One child was excluded as the fetal MRI suggested congenital diaphragmatic hernia. Data were prospectively entered into a database including antenatal and postnatal findings which were then analyzed. RESULTS: Mean gestational age at the time of diagnosis was 20.8 weeks (range 17-29). Fetal interventions included amnioreduction (n = 2) and fetal thoracocentesis (n = 3) in one child. The mean gestational age at delivery was 38.5 weeks (range 31.6-40.6). None of the mothers underwent termination of pregnancy or spontaneous abortion. All patients underwent postnatal radiological assessment. Of the 24 cases, 5 children were symptomatic in the immediate neonatal period and 19 were asymptomatic. Anatomical surgical resection was undertaken in all symptomatic cases. There was one death in this group due to severe pre-existing pulmonary hypoplasia. Among the 19 asymptomatic cases, 5 children were managed conservatively as the lesions were not significant and the remaining 14 (73%) underwent surgical resection. The mean age at surgery was 11.1 months (range 3.3-34 months). Postnatal pathology confirmed CCAM in all operated cases, except one who had pulmonary sequestration. CONCLUSION: Antenatally diagnosed CCAMs have an excellent prognosis except in children with a large lesion and associated with lung hypoplasia. Postnatal investigations are required in all cases to confirm the diagnosis. Symptomatic CCAMs require immediate surgery in the neonatal period with a good outcome. Asymptomatic CCAMs can be safely operated between 9 and 12 months of age.

Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription.

The retinoblastoma-related protein p107 has been shown to be a regulator of the transcription factor E2F. p107 associates with E2F via its pocket region and represses E2F-dependent transcription. In this study, we provide evidence for a novel interaction between p107 and the transcription factor Sp1. We show that p107 can be found endogenously associated with Sp1 in the extracts of several different cell lines. Moreover, in transient transfection assays, expression of p107 represses Sp1-dependent transcription. This repression of Sp1-dependent transcription does not require the DNA-binding domain of Sp1. Transcription driven by a chimeric protein containing the Ga14 DNA-binding domain and the Sp1 activation domains is inhibited by p107. Interestingly, unlike the repression of E2F-dependent transcription, the repression of Sp1-dependent transcription does not depend on an intact pocket region. We show that distinct regions of p107 are involved in the control of Sp1 and E2F.

Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex.

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells.

The damaged-DNA binding protein DDB consists of two subunits, DDB1 (127 kDa) and DDB2 (48 kDa). Mutations in the DDB2 subunit have been detected in patients suffering from the repair deficiency disease xeroderma pigmentosum (group E). In addition, recent studies suggested a role for DDB2 in global genomic repair. DDB2 also exhibits transcriptional activity. We showed that expression of DDB1 and DDB2 stimulated the activity of the cell cycle regulatory transcription factor E2F1. Here we show that DDB2 is a cell cycle-regulated protein. It is present at a low level in growth-arrested primary fibroblasts, and after release the level peaks at the G(1)/S boundary. The cell cycle regulation of DDB2 involves posttranscriptional mechanisms. Moreover, we find that an inhibitor of 26S proteasome increases the level of DDB2, suggesting that it is regulated by the ubiquitin-proteasome pathway. Our previous study indicated that the cullin family protein Cul-4A associates with the DDB2 subunit. Because cullins are involved in the ubiquitin-proteasome pathway, we investigated the role of Cul-4A in regulating DDB2. Here we show that DDB2 is a specific target of Cul-4A. Coexpression of Cul-4A, but not Cul-1 or other highly related cullins, increases the ubiquitination and the decay rate of DDB2. A naturally occurring mutant of DDB2 (2RO), which does not bind Cul-4A, is not affected by coexpression of Cul-4A. Studies presented here identify a specific function of the Cul-4A gene, which is amplified and overexpressed in breast cancers.

Identification of a 60-kilodalton Rb-binding protein, RBP60, that allows the Rb-E2F complex to bind DNA.

Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb.

DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1.

The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, we looked for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. Here we show that DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. We show that the UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. Moreover, the 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1 depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts resulted in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also revealed a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, was enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. We also observed evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulated E2F1-activated transcription. In addition, the transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 was stimulated by coexpression of DDB. Moreover, DDB expression could overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. We speculate that the damaged-DNA binding function of DDB, besides repair, might serve as a negative regulator of E2F1-activated transcription, as damaged DNA will sequester DDB and make it unavailable for E2F1. Furthermore, the binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA.

The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding.

The promoter-specific factor E2F interacts with critical regulatory sequences within the adenovirus E2 promoter. In addition, the level of active factor increases markedly during a virus infection, dependent on E1A function and coincident with the trans activation of E2 transcription. We have purified the E2F factor through a combination of standard biochemical procedures and DNA affinity chromatography. The purified factor was a single polypeptide of 54,000 molecular weight, as determined by UV crosslinking and renaturation of gel-fractionated protein. Addition of affinity-purified factor to an in vitro transcription system resulted in stimulation of transcription from a promoter containing two E2F-binding sites but not promoters lacking binding sites. We thus conclude that E2F is indeed capable of stimulating transcription once it has bound to the promoter.

p21 Disrupts the interaction between cdk2 and the E2F-p130 complex.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription.

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.

p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Both Rb and E7 are regulated by the ubiquitin proteasome pathway in HPV-containing cervical tumor cells.

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitin-proteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated specifically by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome significantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a significant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells.

HPV16 E7 oncoprotein induces expression of a 110 kDa heat shock protein.

Heat shock protein genes are induced by various kinds of stress. Besides stress, the heat shock family gene hsp70 has been shown to be induced by growth-stimulating agents such as the DNA virus oncoproteins and serum. Here, we report cloning of a novel cDNA that encodes a 100 kDa heat shock protein-related polypeptide as a human papillomavirus oncoprotein E7-inducible gene. E7 induces expression of this heat shock protein at the level of RNA synthesis. Moreover, the induction of this heat shock protein-mRNA was dependent on the conserved region 2 of the E7 protein, which is essential for binding to the proteins of the retinoblastoma family.

Site-directed mutant p21 proteins defective in both inhibition of E2F-regulated transcription and disruption of E2F-p130-cyclin-cdk2 complexes.

P21 is a regulatory protein that can contribute to cell cycle arrest by inhibiting the cyclin-dependent-kinases (cdks). However, the mechanism that links the inhibition of the cdk activities and the cell cycle arrest is not well established. To investigate this, we studied a purified endogenous cellular complex which contained E2F (in the form of E2F-4), p130, cyclin, and cdk2. This complex of E2F-p130-cyclin-cdk2 is found mainly in cycling cells and is postulated to be an intermediate that leads to the activation of E2F. We previously showed that p21 could disrupt this complex leading to the accumulation of an E2F-p130 complex and the inhibition of E2F-regulated transcription. We analyzed a group of p21 mutants including those that harbored changes in cyclin- and cdk2-binding motifs. We show that both the cyclin and cdk2 binding motifs of p21 are crucial for the disruption of this endogenous complex of E2F-p130-cyclin-cdk2. This suggests a model where the ability of p21 to inhibit the function of this complex is dependent on interactions with both cyclin and cdk2 molecules. This was substantiated by studies with intact cells. P21 mutants that are impaired in their ability to disrupt the cellular E2F-p130-cyclin-cdk2 complex are also shown to be maximally impaired in the ability to repress E2F-regulated transcription.

The p48 subunit of the damaged-DNA binding protein DDB associates with the CBP/p300 family of histone acetyltransferase.

DDB has been implicated in DNA repair as well as transcription. Mutations in DDB have been correlated with the repair-deficiency disease, xeroderma pigmentosum group E (XP-E). The XP-E cells exhibit deficiencies in global genomic repair, suggesting a role for DDB in that process. DDB also possesses a transcription stimulatory activity. We showed that DDB could function as a transcriptional partner of E2F1. But the mechanism by which DDB stimulates E2F-regulated transcription or carry out its DNA repair function is not understood. To investigate the mechanisms, we looked for nuclear proteins that interact with DDB. Here we show that DDB associates with the CBP/p300 family of proteins, in vivo and in vitro. We suggest that DDB participates in global genomic repair by recruiting CBP/p300 to the damaged-chromatin. It is possible that the histone acetyltransferase activities of the CBP/p300 proteins induce chromatin remodeling at the damaged-sites to allow recruitment of the repair complexes. The observation offers insights into both transcription and repair functions of DDB.

Seatbelt: a double-edged sword.

Intra-abdominal vascular injury due to blunt trauma is unusual in children. Due to its rarity, detailed reports dealing with its management are scarce in paediatric literature. Diagnosis of these injuries is challenging, and a high degree of awareness is necessary for rapid identification and treatment of these injuries. We report the case of a child with seatbelt sign and mesenteric vein injury due to blunt trauma to the abdomen during a motor vehicle accident where the seatbelt was incorrectly placed. She also sustained cervical vertebral injury. The pattern of injuries in children in these situations may differ from that found in adults. While seatbelts have undoubtedly saved many lives, awareness about correct placement of these restraints is extremely necessary.

ARF-induced downregulation of Mip130/LIN-9 protein levels mediates a positive feedback that leads to increased expression of p16Ink4a and p19Arf.

The ARF-MDM2-p53 pathway constitutes one of the most important mechanisms of surveillance against oncogenic transformation, and its inactivation occurs in a large proportion of cancers. Here, we show that ARF regulates Mip130/LIN-9 by inducing its translocation to the nucleolus and decreasing the expression of the Mip130/LIN-9 protein through a post-transcriptional mechanism. The knockdown of Mip130/LIN-9 in p53(-/-) and Arf(-/-) mouse embryonic fibroblasts (MEFs) mimics some effects of ARF, such as the downregulation of B-Myb, impaired induction of G2/M genes, and a decrease in cell proliferation. Importantly, although the knockdown of Mip130/LIN-9 reduced the proliferation of p53 or Arf-null MEFs, only p53(-/-) MEFs showed a senescence-like state and an increase in the expression of Arf and p16. Interestingly, the increase in p16 and ARF is indirect because the Mip130/LIN-9 knockdown decreased the transcription of negative regulators of the Ink4a/Arf locus, such as BUBR1 and CDC6. Chromatin immunoprecipitation assays also reveal that Mip130/LIN-9 occupies the promoters of the BubR1 and cdc6 genes, suggesting that Mip130/LIN-9 is necessary for the expression of these genes. Altogether, these results indicate that there is a feedback mechanism between ARF and Mip130/LIN-9 in which either the increase of ARF or the decrease in Mip130/LIN-9 causes a further increase in the expression of Arf and p16.

Approaches in type 1 diabetes research: A status report.

Type 1 diabetes is a multifactorial disease with an early age of onset, in which the insulin producing beta cell of the pancreas are destroyed because of autoimmunity. It is the second most common chronic disease in children and account for 5% to 10% of all diagnosed cases of diabetes. India is having an incidence of 10.6 cases/year/100,000, and recent studies indicate that the prevalence of type 1 diabetes in India is increasing. However in view of poor health care network, there is no monitoring system in the country. Of the 18 genomic intervals implicated for the risk to develop type 1 diabetes, the major histocompatibility complex (MHC) region on chromosome 6p21.31 has been the major contributor estimated to account for 40-50%, followed by 10% frequency of INS-VNTR at 5' flanking region of the insulin gene on chromosome 11p15.5. However, population studies suggest that > 95% of type 1 diabetes have HLA-DR3 or DR4, or both, and in family studies, sibling pairs affected with type 1 diabetes have a non-random distribution of shared HLA haplotypes. As predisposing genetic factors such as HLA alleles are known, immunological interventions to prevent type 1 diabetes are of great interest. In the present study we have reviewed the status of molecular genetics of the disease and the approaches that need to be adopted in terms of developing patient and suitable control cohorts in the country.