Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein.

BACKGROUND: Long Interspersed Element 1 (LINE-1) is a retrotransposon that is present in 500,000 copies in the human genome. Along with Alu and SVA elements, these three retrotransposons account for more than a third of the human genome sequence. These mobile elements are able to copy themselves within the genome via an RNA intermediate, a process that can promote genome instability. LINE-1 encodes two proteins, ORF1p and ORF2p. Association of ORF1p, ORF2p and a full-length L1 mRNA in a ribonucleoprotein (RNP) particle, L1 RNP, is required for L1 retrotransposition. Previous studies have suggested that fusion of a tag to L1 proteins can interfere with L1 retrotransposition. RESULTS: Using antibodies detecting untagged human ORF1p, western blot analysis and manipulation of ORF1 sequence and length, we have identified a set of charged amino acids in the C-terminal region of ORF1p that are important in determining its subcellular localization. Mutation of 7 non-identical lysine residues is sufficient to make the resulting ORF1p to be predominantly cytoplasmic, demonstrating intrinsic redundancy of this requirement. These residues are also necessary for ORF1p to retain its association with KPNA2 nuclear pore protein. We demonstrate that this interaction is significantly reduced by RNase treatment. Using co-IP, we have also determined that human ORF1p associates with all members of the KPNA subfamily. CONCLUSIONS: The prediction of NLS sequences suggested that specific sequences within ORF1p could be responsible for its subcellular localization by interacting with nuclear binding proteins. We have found that multiple charged amino acids in the C-terminus of ORF1p are involved in ORF1 subcellular localization and interaction with KPNA2 nuclear pore protein. Our data demonstrate that different amino acids can be mutated to have the same phenotypic effect on ORF1p subcellular localization, demonstrating that the net number of charged residues or protein structure, rather than their specific location, is important for the ORF1p nuclear localization. We also identified that human ORF1p interacts with all members of the KPNA family of proteins and that multiple KPNA family genes are expressed in human cell lines.

The impact of multiple splice sites in human L1 elements.

LINE-1 elements represent a significant proportion of mammalian genomes. The impact of their activity on the structure and function of the host genomes has been recognized from the time of their discovery as an endogenous source of insertional mutagenesis. L1 elements contain numerous functional internal polyadenylation signals and splice sites that generate a variety of processed L1 transcripts. These sites are also reported to contribute to the generation of hybrid transcripts between L1 elements and host genes. Using northern blot analysis we demonstrate that L1 splicing, but not L1 polyadenylation, is delayed during the course of L1 expression. L1 splicing can also be negatively regulated by EBV SM protein known to alter this process. These results suggest a potential for L1 mRNA processing to be regulated in a tissue- and/or development-specific manner. The delay in L1 splicing may also serve to protect host genes from the excessive burden of L1 interference with their normal expression via aberrant splicing.

Cis-acting influences on Alu RNA levels.

The human short interspersed repeated element (SINE), Alu, amplifies through a poorly understood RNA-mediated mechanism, termed retroposition. There are over one million copies of Alu per haploid human genome. The copies show some internal variations in sequence and are very heterogeneous in chromosomal environment. However, very few Alu elements actively amplify. The amplification rate has decreased greatly in the last 40 million years. Factors influencing Alu transcription would directly affect an element's retroposition capability. Therefore, we evaluated several features that might influence expression from individual Alu elements. The influence of various internal sequence variations and 3' unique flanks on full-length Alu RNA steady-state levels was determined. Alu subfamily diagnostic mutations do not significantly alter the amount of Alu RNA observed. However, sequences containing random mutations throughout the right half of selected genomic Alu elements altered Alu RNA steady-state levels in cultured cells. In addition, sequence variations at the 3' unique end of the transcript also significantly altered the Alu RNA levels. In general, sequence mutations and 3' end sequences contribute to Alu RNA levels, suggesting that the master Alu element(s) have a multitude of individual differences that collectively gives them a selective advantage over other Alu elements.

Human retroelements may introduce intragenic polyadenylation signals.

In the human genome, the insertion of LINE-1 and Alu elements can affect genes by sequence disruption, and by the introduction of elements that modulate the gene's expression. One of the modulating sequences retroelements may contribute is the canonical polyadenylation signal (pA), AATAAA. L1 elements include these within their own sequence and AATAAA sequences are commonly created in the A-rich tails of both SINEs and LINEs. Computational analysis of 34 genes randomly retrieved from the human genome draft sequence reveals an orientation bias, reflected as a lower number of L1s and Alus containing the pA in the same orientation as the gene. Experimental studies of Alu-based pA sequences when placed in pol II or pol III transcripts suggest that the signal is very weak, or often not used at all. Because the pA signal is highly affected by the surrounding sequence, it is likely that the Alu constructs evaluated did not provide the required recognition signals to the polyadenylation machinery. Although the effect of pA signals contributed by Alus is individually weak, the observed reduction of "sense" oriented pA-containing L1 and Alu elements within genes reflects that even a modest influence causes a change in evolutionary pressure, sufficient to create the biased distribution.

Model structure of the Omp alpha rod, a parallel four-stranded coiled coil from the hyperthermophilic eubacterium Thermotoga maritima.

Omp alpha is an outer-membrane protein that spans the periplasmic space of the hyperthermophilic eubacterium Thermotoga maritima. The molecule contains a globular head with an apparent diameter of 8 nm and a rod-shaped tail of 40 nm length. The sequence of the globular domain is homologous to a conserved region of cell wall-bound proteins and probably attaches Omp alpha to the peptidoglycan. The sequence of the rod domain resembles that of coiled coil proteins and ends in a transmembrane segment that anchors Omp alpha to the outer membrane. We have analysed Omp alpha by scanning transmission electron microscopy (STEM) and by statistical sequence analysis methods. The Omp alpha rod is a tetramer with an unusual periodicity of hydrophobic residues close to 3.6 that differs from the 3.5 periodicity of canonical coiled coils. This is due to periodic omissions of three residues in the heptad repeat pattern ("stutters") whose effect is to locally distort the packing of hydrophobic layers in the core of the coiled coil. Residues in position alpha are shifted to occupy a position halfway between positions alpha and d (x layers) and residues in positions d and e are shifted so that both participate in core packing interactions (da layers). Such distorted layers are frequently found in helical bundles and are characteristic of helices that do not undergo supercoiling. The only homo-oligomeric coiled coil of known structure which contains x and da layers is the three-stranded coiled coil of influenza haemagglutinin. Using geometric constraints derived from this structure, we have built a model for the Omp alpha rod in which the helices have a crossing angle of less than 15 degrees and maintain a residual degree of supercoiling with a pitch of approximately 40 nm. Our analysis of distorted layers in the hydrophobic core of coiled coils and helical bundles shows that stutters must not be viewed as discontinuities but rather as a departure from the canonical "knobs-into-holes" packing that allows helices to interact at a low angle without supercoiling. Although stutters have been considered to weaken helical interactions, their occurrence in a rigid, highly thermostable coiled coil indicates that this may not be generally true. Our analysis also indicates that skips and stutters are two different conventions for describing the same underlying structural feature.

Large-scale analysis of the Alu Ya5 and Yb8 subfamilies and their contribution to human genomic diversity.

We have utilized computational biology to screen GenBank for the presence of recently integrated Ya5 and Yb8 Alu family members. Our analysis identified 2640 Ya5 Alu family members and 1852 Yb8 Alu family members from the draft sequence of the human genome. We selected a set of 475 of these elements for detailed analyses. Analysis of the DNA sequences from the individual Alu elements revealed a low level of random mutations within both subfamilies consistent with the recent origin of these elements within the human genome. Polymerase chain reaction assays were used to determine the phylogenetic distribution and human genomic variation associated with each Alu repeat. Over 99 % of the Ya5 and Yb8 Alu family members were restricted to the human genome and absent from orthologous positions within the genomes of several non-human primates, confirming the recent origin of these Alu subfamilies in the human genome. Approximately 1 % of the analyzed Ya5 and Yb8 Alu family members had integrated into previously undefined repeated regions of the human genome. Analysis of mosaic Yb8 elements suggests gene conversion played an important role in generating sequence diversity among these elements. Of the 475 evaluated elements, a total of 106 of the Ya5 and Yb8 Alu family members were polymorphic for insertion presence/absence within the genomes of a diverse array of human populations. The newly identified Alu insertion polymorphisms will be useful tools for the study of human genomic diversity.

Alu insertion polymorphisms for the study of human genomic diversity.

Genomic database mining has been a very useful aid in the identification and retrieval of recently integrated Alu elements from the human genome. We analyzed Alu elements retrieved from the GenBank database and identified two new Alu subfamilies, Alu Yb9 and Alu Yc2, and further characterized Yc1 subfamily members. Some members of each of the three subfamilies have inserted in the human genome so recently that about a one-third of the analyzed elements are polymorphic for the presence/absence of the Alu repeat in diverse human populations. These newly identified Alu insertion polymorphisms will serve as identical-by-descent genetic markers for the study of human evolution and forensics. Three previously classified Alu Y elements linked with disease belong to the Yc1 subfamily, supporting the retroposition potential of this subfamily and demonstrating that the Alu Y subfamily currently has a very low amplification rate in the human genome.

Characterization of the hormone binding site of natriuretic peptide receptor-C.

The hormone binding site of rat and human natriuretic peptide clearance receptor (NPR-C), a single transmembrane receptor, has been further refined by mutagenesis. In addition to residue 188 (rat Ala, human Ile), which completely inverts the pharmacology of the rat and human receptors [Engel et al. (1994) J. Biol. Chem. 269, 17005-17008], we report a second key residue at position 205 (rat Tyr, human Asn) which modulates affinity to a limited number of ligands. Orthologous mutation of both residues results in tighter binding for human and weaker binding for rat NPR-C. The ligand binding fold of the receptor is formed by at least the first half of the extracellular domain containing two intramolecular disulfide loops, with the two affinity-modulating residues 188 and 205 in the second loop.

Hepatitis G virus RNA and hepatitis G virus antibodies in renal transplant recipients: prevalence and risk factors.

BACKGROUND: Hepatitis G virus (HGV/GBV-C) RNA indicating current infection has been frequently isolated from the sera of transplant recipients and other multitransfused individuals. Lifetime exposure to the virus, however, is unknown. We carried out a study to determine the prevalence and risk factors of HGV antibodies and of HGV RNA among renal transplant recipients, and to investigate possible associations between HGV RNA and immunosuppressive treatment. METHODS: HGV RNA was detected by reverse transcriptase-polymerase chain reaction, and HGV antibodies (anti-E2) by a newly developed immunoassay. To assess risk factors for HGV exposure, univariate and multivariate analysis was performed. RESULTS: Of the 221 patients, 14% were HGV RNA positive and 40% had HGV antibodies. Both HGV RNA and anti-HGV were present in only two individuals. Thus, the overall HGV exposure prevalence was 53%. It increased significantly with the number of blood transfusions. In logistic regression, the adjusted HGV exposure prevalence odds ratio was 5.7 (95% confidence interval [CI]: 2.2-15) among patients with > or =10 transfusions (baseline: no transfusions). Other independent risk factors were a longer duration of hemodialysis and a longer time interval since transplantation. HGV viremia was not associated with the type of immunosuppressive treatment. Alanine aminotransferase levels were not significantly increased among HGV RNA-positive patients. CONCLUSIONS: Much higher proportions of renal transplant recipients were exposed to HGV than is suggested by HGV RNA detection alone. The majority of infected individuals apparently eliminate the virus over time. Contaminated blood transfusions have to be regarded as a main risk factor for HGV infection.

High MHC class I expression correlates with slow growth in UV-induced skin carcinomas in hairless mice.

An experiment was set up to investigate the relationship, if any, between cell surface MHC class I expression and the growth rate for skin tumors induced by two different UV radiation regimens in hairless mice. Two groups of 20 hairless mice were each irradiated with either a UVA radiation source (2 SED per session) or broad-spectrum UV radiation (UVB) (8.1 SED per session) 5 days a week during the entire experiment. In the UVA group, 17 out of 20 animals developed tumors, and 10 of these grew to a diameter of > or = 5 mm. In the UVB group, 19 out of 20 animals developed tumors, and 15 of these grew to a diameter of > or = 5 mm. The tumor induction time, i.e. the time from the start of UV treatment to tumor appearance, was found to be significantly longer (p<0.01) in the UVA than in the UVB group. This is in accordance with previous findings. Of the 25 tumors growing to a diameter of > or = 5 mm, 11 were established as cultured cell lines (4 UVA and 7 UVB tumors). These uncloned cell lines were analyzed for surface expression of major histocompatibility complex class I by FACS analysis. There was a clear correlation between high MHC class I expression and slow growth of the individual tumors (p<0.05). This suggests a role for the MHC class I governed, i.e. cytotoxic T-cell-mediated, reactions in deciding the fate of UV-induced skin cancers. No correlation was found between MHC class I expression and tumor induction time.

Tumors developing in nude mice express unusually large amounts of MHC class I antigens.

Tumors were induced in athymic, T-cell-deficient nude mice and in syngeneic normal haired mice by treatment with low doses of 3-methylcholantrene (MCA). The tumors were studied for tumor cell expression of MHC class I molecules and for immunogenicity by transplantation to syngeneic haired recipients. Ten tumors were obtained by the MCA treatment, six from nude and four from haired mice. They were all fibrosarcomas as judged from their microscopic appearance. Five of the "nude" tumors expressed measurable amounts of MHC class I molecules and two of them expressed high amounts. Both were immunogenic in the sense that they evoked a cytotoxic T-cell response in transplanted haired recipients. Only one of the four "haired" tumors expressed measurable amounts of MHC class I, and none of them were immunogenic. These findings support the concept that some tumors are immunoselected at an early point of time in their existence in a host with a normal immune system and that this results in an elimination of tumor cell variants which are highly immunogenic for the T-cell system, leaving the low or non-immunogenic variants. These take over and grow and kill their host. The results suggest that tumor cell variants expressing high amounts of MHC class I are important targets in the immunoselection in hosts with a normal immune system.

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T-cell-mediated defense mechanisms may confer upon the bearer a lower resistance to 3-methylcholanthrene and a different MHC profile of the ensuing tumor.

Expression of beta 2-microglobulin by premalignant epithelium.

Many human tumors express low amounts of HLA class I molecules relative to the normal cells from which they are derived. From experimental work it is clear that the malignant behavior of a tumor cell may depend on its MHC class I expression. Therefore, it is of obvious interest to study the HLA class I expression of human tumors in their various stages. We have studied the HLA class I expression by the cells in premalignant epithelial lesions and invasive carcinoma of the bladder and uterine cervix using immunoperoxidase staining for beta 2-microglobulin of paraffin-embedded tissue. We here assume that beta 2-microglobulin expression by malignant and premalignant cells equals HLA class I expression. Thirty-two of the 36 invasive tumors expressed less overall beta 2-microglobulin than cells from the normal epithelium. In contrast, approximately two-thirds of 34 premalignant bladder epithelia and 47 premalignant cervix epithelia displayed higher overall beta 2-microglobulin expression than the normal epithelium. Thus, a systematic large-scale elimination of HLA class I high-expressing tumor cell variants may take place only after the tumor penetrates the basement membrane.

The role of cytotoxic T-lymphocytes in the prevention and immune surveillance of tumors--lessons from normal and immunodeficient mice.

The idea of immunological surveillance against cancer has existed for nearly 100 years but as no conclusive evidence has yet been published the importance of the cellular immune defense in the detection and removal of incipient or existing tumors is still a hotly debated subject. However, in order to select a relevant immunotherapeutic strategy in the treatment of cancer, a fundamental understanding of the basic immunologic conditions under which a tumor develops and exists is a prerequisite. Therefore, a murine model was set up that we hoped would enable us to confirm or reject the theory of immunological surveillance. A large panel of methylcholanthrene induced tumors was established in T-cell immunodeficient nude mice and congenic normal mice to study the influence of the immune system on developing tumors. As nude mice developed tumors fastest and with the highest incidence, we concluded that in this model the immune system constituted a 'tumor-suppressive factor' delaying and sometimes abrogating tumor growth, i.e. performing immune surveillance. Immunogenicity of the tumors was assessed by transplantation back to normal histocompatible mice. Tumors originating from the immunodeficient nude mice turned out to be far more immunogenic than tumors from normal mice, resulting in a high rejection rate. CD8+ cytotoxic T cells were found to be indispensable for this rejection, leading to the conclusion that the cytotoxic T cells perform immune selection in normal mice, eliminating immunogenic tumor cell variants in the incipient tumor. In this review, we discuss the difficulties facing immunotherapy when conclusions are drawn from the presented observations and hypotheses.

Confocal laser scanning microscopy of tumor/vessel relationship in xenografts in nude and scid mice.

Using confocal laser scanning microscopy we studied sections of the T24B, a human bladder carcinoma, grown in C.B.-17 scid/scid or NMRI nu/nu mice in order to examine the relationship between tumor tissue and tumor vessels. Tumor cells were labelled with FITC-anti-cytokeratin and blood vessel endothelia with Cy3-labelled BS-I lectin. In contrast to our expectation, no major leaks in the endothelial lining of blood vessels were observed. We are looking for a suitable marker for mouse lymphatics in order to investigate their possible role.

Synthesis and anti-angiogenic effect of conjugates between serum albumin and non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.

Natural inhibitors and fibrinogen/fibrin degradation products in patients with myocardial infarction.

The activity of the inhibitor of activated Stuart factor (anti-Xa) was evaluated together with the activity of the antiactivator of plasminogen (antiplasminogen) and the amount and nature of the fibrinogen/fibrin degradation products (FDP), as indicators of the existence of the risk for hypercoagulability in patients with acute and recent myocardial infarction (AMI and RMI, respectively). The activity of anti-Xa was diminished in the AMI group with a p less than 0.001. The antiplasminogen activity was increased in both groups with a p less than 0.001; on the other hand, the amount of FDP was increased in the AMI patients and they were of the 'early' type, while in the RMI group the increase was not as marked, but the FDP were of the 'late' type. In both groups the euglobulin lysis time was very prolonged, while the plasminogen did not vary significantly. The tests described appear to be valuable tools for studying the status of the cardiovascular system during cardiac rehabilitation.

Salmeterol in two different doses in the treatment of nocturnal bronchial asthma poorly controlled by other therapies.

This study compared the efficacy of inhaled salmeterol (SM) 50 micrograms twice a day with SM 100 micrograms once a day (at night) and placebo in patients with poorly controlled nocturnal asthma despite treatment with corticosteroids, oral long-acting bronchodilators, or both. This was a two-center, double-blind, randomized, crossover study with three treatment periods, each of 3 weeks' duration. The first treatment period was preceded by two 1-week run-in periods, and the last treatment period was followed by a 2-week follow-up period. In the three treatment periods, patients received either SM 50 micrograms twice a day, SM 100 micrograms nightly, or placebo, in randomized order. Salbutamol metered-dose inhaler was given as relief medication. Of the 41 randomized patients, 38 were evaluable for more than one treatment period. Efficacy and safety were determined by daily record card data: morning and evening peak expiratory flow rates (PEFR), daytime and nighttime asthma symptom scores, and rescue salbutamol use. At clinic visits, FEV1 and FVC were measured, and the physicians' and the patients' respective assessments of the study mediation were noted. The study showed that the two doses, SM 50 micrograms twice a day and SM 100 micrograms nightly, were equally effective in controlling nocturnal asthma symptoms and were significantly better than placebo. SM was well tolerated, and no unexpected problems were revealed. The adverse events reported during this study were related either to the patient's underlying disease or to an intercurrent respiratory infection.