UVA, UVB and incidence of cutaneous malignant melanoma in Norway and Sweden

Latitudinal dependencies of UVA and UVB were studied together with relevant epidemiological data for squamous cell carcinoma (SCC) and cutaneous malignant melanoma (CMM) in Norway and Sweden. Our data support the hypothesis that solar UVA radiation may play a role for CMM induction. The etiologies of SCC and CMM are different according to a latitudinal dependency and differences in age curves. Sun exposure patterns, age-related decay rates of repair of UV damage and sex hormones may play different roles for the two skin cancers. Also, UVB induction of vitamin D may be involved. CMM incidence rates among young people have decreased or been constant since about 1990 in Norway and Sweden. All reasons for UVA contributing to CMM will be discussed.     

Energy trapping and detrapping in reaction center mutants from Rhodobacter sphaeroides

Time-resolved fluorescence of chromatophores isolated from strains of Rhodobacter sphaeroides containing light harvesting complex I (LHI) and reaction center (RC) (no light harvesting complex II) was measured at several temperatures between 295 K and 10 K. Measurements were performed to investigate energy trapping from LHI to the RC in RC mutants that have a P/P(+) midpoint potential either above or below wild-type (WT). Six different strains were investigated: WT + LHI, four mutants with altered RC P/P(+) midpoint potentials, and an LHI-only strain. In the mutants with the highest P/P(+) midpoint potentials, the electron transfer rate decreases significantly, and at low temperatures it is possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both complexes. In all mutants, fluorescence kinetics are multiexponential. To explain this, RC + LHI fluorescence kinetics were analyzed using target analysis in which specific kinetic models were compared. The kinetics at all temperatures can be well described with a model which accounts for the energy transfer between LHI and the RC and also includes the relaxation of the charge separated state P(+)H(A)(-), created in the RC as a result of the primary charge separation.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

Targeting of nebulin fragments to the cardiac sarcomere

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.     

Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3′→5′ RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3′-end.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

Linker region of nebulin family members plays an important role in targeting these molecules to cellular structures

The nebulin family of actin-binding proteins plays an essential role in cytoskeletal dynamics and actin filament stability. All of the family members are modular proteins with their key defining structural feature being the presence of the 35-residue nebulin modules. The family members now include nebulin, nebulette, N-RAP, LASP-1, and LIM-nebulette. Nebulin and nebulette are associated with the thin filament/Z-line junction of striated muscle. LASP-1 and LIM-nebulette are found within focal adhesions, and N-RAP is associated with muscle cellular junctions. Although much investigation has focused on the role of the interactions between nebulin modules and actin, each of these proteins contains other domains that are essential for their cellular targeting and functions. The serine-rich linker region of nebulette has previously been shown to serve just such a purpose by targeting the association of the nebulin modules to the cardiac Z-line in cultured cardiomyocytes. In this report, we analyze the targeting functions of the homologous regions of LASP-1 and LIM-nebulette in their incorporation into focal adhesions. We have found that the linker region of LASP-1 is indeed important for its cellular localization and that the shortened linker region of LIM-nebulette drives the association of nebulin modules to focal adhesions. This work was supported by grants from the National Institutes of Health-HLB and the National Council of the American Heart Association to C.L.M.     

Considering specific clinical features as evidence of pathogenic copy number variants

Since the introduction of high-resolution microarray technologies, it has become apparent that structural chromosomal rearrangements can lead to a wide variety of clinical manifestations, including developmental delay/intellectual disability (DD/ID). It has been shown previously that the diagnostic yield of genome-wide array-based identification of submicroscopic alterations in patients with ID varies widely and depends on the patient selection criteria. More attempts have recently been made to define the phenotypic clues of pathogenic copy number variants (CNVs). The aim of this study was to investigate a well-phenotyped cohort of patients with DD/ID and determine whether certain clinical features may serve as indicators for pathogenic CNVs. A retrospective analysis was conducted for patients with DD/ID (n?=?211) who were tested using genome-wide chromosomal microarray technologies and a review of the clinical data was performed. Pathogenic CNVs were detected in 29 patients. In comparison with individuals who had normal molecular karyotyping results (n?=?182), malformations of the musculoskeletal system; congenital malformations of the CNS (particularly hydrocephalus and congenital malformations of the corpus callosum); minor anomalies of the eye, face, and neck subgroup (particularly downward-slanting palpebral fissures, minor anomalies of the ear, and micrognathia); brachydactyly; and umbilical hernia were more common in patients with chromosomal alterations. A multivariate logistic regression analysis allowed the identification of three independent pathogenic CNV predictors: congenital malformations of the corpus callosum, minor anomalies of the ear, and brachydactyly. Insights into the chromosomal phenotype may help to increase the diagnostic yield of microarray technologies and sharpen the distinction between chromosomal alterations and other conditions.