Structure and anomalous underscreening in ethylammonium nitrate solutions confined between two mica surfaces.

The observation of long-range interactions across ionic liquids and highly concentrated electrolytes, extending far beyond the Debye-Hückel prediction and beyond the range predicted in liquid state theory, has been called 'anomalous underscreening'. A number of theoretical and experimental works have explored this phenomenon over recent years, although its origin is not yet fully understood. Most of the experimental studies of anomalous underscreening until now involved aprotic ionic liquids, and so it is of interest to explore interactions in protic ionic liquids where the distribution of charge in the fluid is different in nature. Here we present direct measurements of the interaction force as a function of separation distance, measured using a surface force balance, across solutions of a protic ionic liquid ethylammonium nitrate (EAN) and its mixtures with water over a range of volume fractions from 10 vol% to 100 vol% EAN. The results reveal intricate details about near-surface ordering and dynamics at the EAN-mica interface as well as anomalous underscreening consistent with that observed in the past with aprotic ionic liquids.

Plasmapheresis to treat Osmotic Demyelination Syndrome from overly rapid plasma sodium correction.

Osmotic demyelination syndrome results from overly rapid serum sodium correction and is often iatrogenic. We report a 50-year-old hypertensive woman on Indapamide presenting with malaise, dizziness and serum sodium less than 100mmol/l who developed osmotic demyelination syndrome after correction of the hyponatremia. Good neurological recovery was seen after plasmapheresis.

Rescue effects: irradiated cells helped by unirradiated bystander cells.

The rescue effect describes the phenomenon where irradiated cells or organisms derive benefits from the feedback signals sent from the bystander unirradiated cells or organisms. An example of the benefit is the mitigation of radiation-induced DNA damages in the irradiated cells. The rescue effect can compromise the efficacy of radioimmunotherapy (RIT) (and actually all radiotherapy). In this paper, the discovery and subsequent confirmation studies on the rescue effect were reviewed. The mechanisms and the chemical messengers responsible for the rescue effect studied to date were summarized. The rescue effect between irradiated and bystander unirradiated zebrafish embryos in vivo sharing the same medium was also described. In the discussion section, the mechanism proposed for the rescue effect involving activation of the nuclear factor κB (NF-κB) pathway was scrutinized. This mechanism could explain the promotion of cellular survival and correct repair of DNA damage, dependence on cyclic adenosine monophosphate (cAMP) and modulation of intracellular reactive oxygen species (ROS) level in irradiated cells. Exploitation of the NF-κB pathway to improve the effectiveness of RIT was proposed. Finally, the possibility of using zebrafish embryos as the model to study the efficacy of RIT in treating solid tumors was also discussed.

Structural rearrangement of the retinoblastoma gene in human breast carcinoma.

Structural changes of the human retinoblastoma gene have been demonstrated previously in retinoblastoma and some clinically related tumors including osteosarcoma. Structural aberrations of the retinoblastoma locus (RB1) were observed in 25% of breast tumor cell lines studied and 7% of the primary tumors. These changes include homozygous internal deletions and total deletion of RB1; a duplication of an exon was observed in one of the cell lines. In all cases, structural changes either resulted in the absence or truncation of the RB1 transcript. No obvious defect in RB1 was detected by DNA blot analysis in primary tumors or cell lines from Wilms' tumor, cervical carcinoma, or hepatoma. These results further support the concept that the human RB1 gene has pleiotropic effects on specific types of cancer.

Structural evidence for the authenticity of the human retinoblastoma gene.

The retinoblastoma (Rb) gene is the prototype for a class of recessive human cancer genes in which loss of activity of both normal alleles is thought to be associated with tumorigenesis. Sixteen of 40 retinoblastomas examined with a complementary DNA probe shown to be the Rb gene had identifiable structural changes of the Rb gene including in some cases homozygous internal deletions with corresponding truncated transcripts. An osteosarcoma also had a homozygous internal deletion with a truncated transcript. In addition, possible hot spots for deletion were identified within the Rb genomic locus. Among those tumors with no identifiable structural changes there was either absence of an Rb transcript or abnormal expression of the Rb transcript. Comparison of the structural changes in the tumor cells and fibroblasts of certain patients provided support for Knudson's two-hit hypothesis for the development of retinoblastoma at the molecular level. The ability to detect germline structural deletions in fibroblasts from some patients with bilateral retinoblastoma also indicates that the isolated gene is useful for diagnostic purposes.

Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product.

The human retinoblastoma gene (RB1) encodes a protein (Rb) of 105 kilodaltons that can be phosphorylated. Analysis of Rb metabolism has shown that the protein has a half-life of more than 10 hours and is synthesized at all phases of the cell cycle. Newly synthesized Rb is not extensively phosphorylated (it is "underphosphorylated") in cells in the G0 and G1 phases but is phosphorylated at multiple sites at the G1/S boundary and in S phase. HL-60 cells that were induced to terminally differentiate by various chemicals lost their ability to phosphorylate newly synthesized Rb at multiple sites when cell growth was arrested. These findings suggest that underphosphorylated Rb may restrict cell proliferation.

Identification of protein products encoded by the proto-oncogene int-1.

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.

Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins.

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells.

Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene.

The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.

Functional outcome of the hand following flexor tendon repair at the 'no man's land'.

PURPOSE: To evaluate the functional outcome of the hand following flexor tendon repair at 'no man's land' using 2 strands of a modified Kessler core suture and combined controlled motion rehabilitation protocol. METHODS: Records of 31 zone-2 flexor tendon injuries in 21 digits of 16 patients between July 2000 and June 2005 were reviewed retrospectively. The injured tendons were repaired within 24 hours using 2 strands of a modified Kessler core suture, reinforced by a continuous circumferential epitendon suture. All patients completed a rehabilitation protocol that included active extension against a rubber band, passive flexion, and controlled passive extension and passive flexion exercises. Functional outcome of the fingers was assessed using the Buck-Gramcko II score. Hand grip strength, rehabilitation period, and rupture rate were also measured. RESULTS: 17 (81%) out of 21 digits in 15 out of 16 patients achieved an excellent-to-good functional grade. The remaining patient with concomitant injuries to 4 (19%) digits attained a poor functional grade, attributable to poor compliance with the rehabilitation protocol. The mean rehabilitation period was 130 days and the mean grip strength was 78% that of the uninjured side. Concomitant digital nerve injury did not adversely affect the final outcome. Only one (4.8%) patient experienced a rupture. CONCLUSION: The surgical method and rehabilitation protocol used for zone-2 flexor tendon injury is safe and results in a reasonably good functional outcome.

Microalbuminuria prevalence study in hypertensive type 2 diabetic patients in Malaysia.

Microalbuminuria is the earliest indicator of diabetic kidney disease and generalised vascular endothelial dysfunction. The Microalbuminuria Prevalence (MAP) Study was carried out to assess the prevalence of macroalbuminuria, microalbuminuria and normoalbuminuria in Asian hypertensive patients with type 2 diabetes on usual care. This paper presents a subanalysis of data from patients in Malaysia. In 733 analysed patients, the prevalence of macroalbuminuria and microalbuminuria was 15.7% and 39.7%, respectively. The high prevalence of diabetic nephropathy in these high-risk patients is a cause for concern, and the Malaysian Health Care system should be prepared for a pandemic of end-stage renal disease due to diabetic nephropathy.

Scanning SQUID susceptometers with sub-micron spatial resolution.

Superconducting QUantum Interference Device (SQUID) microscopy has excellent magnetic field sensitivity, but suffers from modest spatial resolution when compared with other scanning probes. This spatial resolution is determined by both the size of the field sensitive area and the spacing between this area and the sample surface. In this paper we describe scanning SQUID susceptometers that achieve sub-micron spatial resolution while retaining a white noise floor flux sensitivity of ≈2µΦ0/Hz1/2. This high spatial resolution is accomplished by deep sub-micron feature sizes, well shielded pickup loops fabricated using a planarized process, and a deep etch step that minimizes the spacing between the sample surface and the SQUID pickup loop. We describe the design, modeling, fabrication, and testing of these sensors. Although sub-micron spatial resolution has been achieved previously in scanning SQUID sensors, our sensors not only achieve high spatial resolution but also have integrated modulation coils for flux feedback, integrated field coils for susceptibility measurements, and batch processing. They are therefore a generally applicable tool for imaging sample magnetization, currents, and susceptibilities with higher spatial resolution than previous susceptometers.

Analysis of viral and cellular gene expression during progression and suppression of the transformed phenotype in type 5 adenovirus-transformed rat embryo cells.

Transformation of secondary Sprague-Dawley rat embryo (RE) cells with type 5 adenovirus (Ad5) results in morphologically transformed cells which can undergo a series of sequential changes resulting in enhanced expression of the transformed phenotype, a process termed progression. Selection for a progressed phenotype often occurs after growth in agar or tumor formation in nude mice, and this process is reversible following treatment of cells with 5-azacytidine. In the present study we have analyzed a series of clonal populations of Ad5-transformed RE cells representing different stages in a defined progression lineage. Progression was not associated with alterations in the steady-state levels of mRNA produced by the viral transforming genes, E1A and E1B, or the cellular gene, c-myc. In addition, the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which induces expression of a progressed phenotype in Ad5-transformed RE cells, did not significantly alter the RNA transcription rates of the Ad5 E1A or E1B genes, the TPA-inducible gene TPA-S1 or the TPA-responsive genes Pro1 or protein kinase C. TPA did, however, increase by 1 h the steady-state level of c-fos mRNA, but this effect was similar in both progressed and unprogressed cells. Progression also did not involve a change in the RNA transcription rate of a number of cellular and viral genes, including actin, c-Ha-ras, c-myc, v-fos, erbB, TGF-alpha, TGF-beta, Pro-2, transin, TPA-R1, v-myb and c-mos, or other adenovirus genes in addition to E1A and E1B, including E2A and E4. Immunoblotting analysis using E1B polyclonal antiserum further indicated that progression was not associated with changes in the levels of an Mr 21,000 polypeptide encoded by E1B. Similarly, immunoprecipitation analysis with an Ad2 E1A monoclonal antibody indicated similar levels of the Mr 55,000 and 48,000 E1A polypeptides, as well as coprecipitated proteins of Mr 300,000, 107,000 and 105,000 [which is the retinoblastoma (Rb) protein], in E11 and E11-NMT cells. Immunoprecipitation of cell lysates with a monoclonal antibody specific for the Mr 105,000 Rb protein further demonstrated that progression also was not associated with a change in the level or state of phosphorylation of the Rb protein. However, transfection of a human Rb gene (also containing a neomycin resistance gene) into Ad5-transformed RE cells was more inhibitory, with respect to formation of G418-resistant colonies, in unprogressed than in progressed Ad5-transformed RE cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Engineered mutants of pRB with improved growth suppression potential.

We have constructed a panel of substitution mutants which affect one or more of the putative cdk target sites of the RB protein. We have examined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can repress E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumor cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the ability to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56RB and a novel mutant of RB containing multiple substitutions near its nuclear localization signal. Both classes of RB mutants have greater activity than the wild-type protein. Because RB is a key regulator of cell cycle progression, expression of a more potent, phosphorylation resistant RB may have utility in both RB(-/-) and RB(+/+) tumors as well as in hyperproliferative disorders.

Altered expression of the retinoblastoma (RB) gene in small-cell carcinoma of the lung.

Nine lung small-cell carcinoma (SCC) cell lines and 9 lung non-SCC cell lines were examined for structural changes of the retinoblastoma (RB) gene as well as its expression using a complementary DNA probe. The RB protein product was investigated using an anti-RB antibody which we produced. Although homozygosity or hemizygosity of the RB gene was suggested in 8 of 9 SCCs and one of 2 large cell carcinomas (LCCs) by Southern blot analysis using an RB cDNA probe and polymorphic DNA markers for chromosome 13, no obvious structural changes of the RB gene were detected in these 18 cell lines. However, RB transcripts were either markedly reduced in quantity or abnormal in length in 3 of 9 SCCs. The specific 115 kD protein was not immunoprecipitated by the anti-RB antibody in all 9 SCCs with either normal or abnormal size RB mRNA. Three of 4 adenocarcinomas (AdCs), all 3 squamous cell carcinomas, and one of 2 LCCs expressed normal size RB mRNA, and the 115 kD protein was immunoprecipitated by the anti-RB antibody. The 115 kD protein was also absent in one of 2 LCCs with shortened RB mRNA and in one of 4 AdCs with low level of RB mRNA expression. These results strongly suggest that inactivation of the RB gene might be involved in the development of lung cancers, especially of SCCs.

The retinoblastoma gene is frequently altered leading to loss of expression in primary breast tumours.

We have analysed the organisation of the retinoblastoma (RB1) gene in 77 primary breast carcinomas, in metastatic tissue derived from 16 of those primary tumours, and in a variety of benign breast lesions. Expression of RB1 was also assessed in most samples by immunohistochemical detection of the RB1 protein in tissue sections. Structural abnormalities to RB1 were detected in DNA from 15/77 (19%) of primary breast carcinomas examined. Where DNA was available from metastatic tissue derived from such primary tumours, the same aberration could be detected. No alterations were seen in benign breast lesions. 16/56 (29%) of tumours examined for expression by immunohistochemical methods showed a proportion of tumour cells to be completely negative for the RB1 protein. All tumours in which a structural alteration to RB1 was detected had a proportion of negative cells, except for one case where all cells were positive. Several primary tumour samples were identified where there was no detectable structural change to the gene, but there was loss of expression in some tumour cells. The data presented here demonstrate that changes to the RB1 gene leading to loss of expression of both alleles are frequent in primary human breast tumours.

The Rb gene suppresses the growth of normal cells.

The suppression of tumor formation, first demonstrated by somatic cell hybrid and microcell fusion experiments, suggests the existence of a class of genes that selectively suppress the growth of tumor cells but not normal cells. The reintroduction of these genes into tumor cells presumably renders the cells responsive to in vivo growth inhibitory environment. As the inheritance of a defective retinoblastoma gene (Rb-1) allele results in a predisposition to the development of various cancers, and since inactivation of both alleles are observed in tumor cells, the Rb gene has been suspected to have the ability to suppress tumor growth. Data presented here demonstrated that different types of normal cells, which have a limited life span, were also growth arrested by a transfected Rb gene. Cell lines which are resistant to the growth suppression effect of the Rb gene in vitro, retain the ability to form tumors in nude mice even in the presence of a stable and highly expressed wild type Rb protein. We conclude that while the Rb gene can suppress the growth of many tumor cell lines, its growth suppression effect is not tumor specific.

Modulation of NF-κB in rescued irradiated cells.

Studies by different groups on the rescue effect, where unirradiated bystander cells mitigated the damages in the irradiated cells, since its discovery by the authors' group in 2011 were first reviewed. The properties of the rescue effect were then examined using a novel experimental set-up to physically separate the rescue signals from the bystander signals. The authors' results showed that the rescue effect was mediated through activation of the nuclear factor-κB (NF-κB) response pathway in the irradiated cells, and that the NF-κB activation inhibitor BAY-11-7082 did not affect the activation of this response pathway in the irradiated cells induced by direct irradiation.