[Diagnostic utility of dermoscopy in pigmented actinic keratosis]. / Utilidad de la dermatoscopia en el diagnóstico de las queratosis actínicas pigmentadas.

The diagnosis of pigmented actinic keratosis can be complicated in clinical practice. The differential diagnosis with lentigo maligna melanoma can be difficult due to common clinical and dermoscopic characteristics. We present 5 cases of pigmented actinic keratosis in 4 patients. The most common dermoscopic finding was a grayish-brown granulation with a perifollicular distribution, present in all lesions, followed by rhomboidal structures in 4 cases, and an annular-granular pattern in 3. In no case were asymmetrical pigmented follicular openings observed. We draw attention to key findings that aid preoperative diagnosis of pigmented actinic keratosis.

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. An examination of steady-state RNA levels in cells carrying these mutations and a comparison with similar data from other dhfr mutants (A. M. Carothers, R. W. Steigerwalt, G. Urlaub, L. A. Chasin, and D. Grunberger, J. Mol. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter.

The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with anti-Rb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both effects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene.

Phosphorylation of glycogen synthase in isolated rabbit hepatocytes.

Specific antibodies were used to purify glycogen synthase from isolated rabbit hepatocytes that had been incubated in a medium containing [32P]phosphate. The enzyme gave rise to two main 32P-labeled CNBr fragments of electrophoretic mobilities similar to those obtained after phosphorylation of the enzyme by individual protein kinases in vitro.

Identification by RNA-based arbitrarily primed PCR of the involvement of cytochrome c oxidase in the development of resistance to methotrexate.

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.

Inactivation of basal glycogen synthase by glucagon and epinephrine in hepatocytes from fed rats.

Glucagon and epinephrine promote the inactivation of basal glycogen synthase in hepatocytes isolated from fed rats. However, this effect is only observable when the activation state of glycogen synthase is measured using the low glucose-6-P/high glucose-6-P activity ratio assay. This inactivation is the consequence of an increase in the kinetic parameters (S0.5 for UDP-glucose and M0.5 for glucose-6-P) of the enzyme. Therefore, this work demonstrates these hormones are also able to control glycogen synthase from fed animals.

Different effects of glucagon and epinephrine on the kinetic properties of liver glycogen synthase.

Kinetic constants of glycogen synthase (M0.5 for glucose-6-P and S0.5 for UDP-glucose) were determined after hepatocytes isolated from starved rats were incubated with either glucagon or epinephrine. Incubation with these hormones resulted in an increase in both S0.5 and M0.5. However, the action of glucagon resulted in great modifications on S0.5 whereas epinephrine affected mainly M0.5. Therefore, glucagon and epinephrine alter the kinetic properties of glycogen synthase provoke the phosphorylation of glycogen synthase at different site(s) acting through different mechanisms.

Glucose has to be phosphorylated to activate glycogen synthase, but not to inactivate glycogen phosphorylase in hepatocytes.

2-Deoxyglucose and 5-thioglucose, in the same fashion as glucose, cause the inactivation of the rat hepatocyte glycogen phosphorylase and the activation of glycogen synthase. However, 6-deoxyglucose and 1,5-anhydroglucitol inactivate phosphorylase without increasing the activation state of glycogen synthase. With 3-O-methylglucose no changes in the activity of these enzymes occurred. These results prove that while glucose is the molecule that triggers the inactivation of phosphorylase, glucose 6-phosphate is the signal for glucose synthase activation and that a metabolite control of the activation state of glycogen synthase is operative in hepatocytes.

Protein kinase C inhibitors reduce phorbol ester-induced resistance to methotrexate in Chinese hamster ovary cells.

Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies surviving methotrexate (MTX) exposure in a dose-dependent manner upon short incubation with Chinese hamster ovary (CHO) cells. Seventy percent of the isolated colonies showed increased copy number for the dihydrofolate reductase gene. EGTA prevents the increase in resistance triggered by TPA. Calcium ionophore A23187 and angiotensin II also increase this resistance, suggesting that calcium is involved in this process. Protein kinase C (PKC) from CHO cells is rapidly activated by TPA, A23187 and angiotensin II. PKC inhibitors, 1-(5-Isoquinolinylsulphonyl)-2-methyl-piperazine (H-7), glycyrrhetinic acid, staurosporine and calphostin C decrease the generation of resistant colonies to MTX upon incubation with TPA. However, 5 nM staurosporine on its own increases resistance to MTX while having the ability to translocate CHO PKC. In vitro, H-7, staurosporine and calphostin C inhibit PKC activity translocated by TPA incubation with CHO cells. We conclude that PKC, the activity of which is dependent on calcium and phospholipids, is part of the pathway that leads to development of increased resistance to MTX. Thus, inhibition of PKC prevents the appearance of this resistance. Our results suggest the possibility of using non-toxic PKC inhibitors as resistance modulators in MTX chemotherapy.

Differential induction of stearoyl-CoA desaturase and acyl-CoA oxidase genes by fibrates in HepG2 cells.

We studied whether two typical effects of fibrates, induction of stearoyl-CoA desaturase (EC 1.14.99.5) and peroxisome proliferation, are related. The effect of bezafibrate on the activity and mRNA of stearoyl-CoA desaturase and acyl-CoA oxidase in the liver and epididymal white adipose tissue of male Sprague-Dawley rats was determined. The same parameters were measured in HepG2 cells, a cell line resistant to peroxisome proliferation, following incubation with ciprofibrate. Bezafibrate increased the hepatic mRNA levels (14.5-fold on day 7) and activity (9.3-fold on day 15) of acyl-CoA oxidase. Stearoyl-CoA desaturase mRNA levels were transiently increased (2.7-fold on day 7), while its activity remained increased at the end of the treatment (2.4-fold). In white adipose tissue, bezafibrate increased the mRNA (5-fold) and activity (1.9-fold) of acyl-CoA oxidase, while stearoyl-CoA desaturase was not modified. Ciprofibrate addition to HepG2 cells cultured in 7% fetal bovine serum (FBS) only increased the stearoyl-CoA desaturase mRNA (1.9-fold). When cells were cultured in 0.5% FBS, ciprofibrate increased acyl-CoA oxidase mRNA (2.2-fold), while the increase in stearoyl-CoA desaturase mRNA was identical (1.9-fold). Further, its activity was also increased (1.5-fold). Incubation of HepG2 cells in the presence of cycloheximide did not alter the capacity of ciprofibrate to induce stearoyl-CoA desaturase mRNA, whereas the presence of actinomycin abolished the induction. In addition, preincubation of HepG2 cells with ciprofibrate increased the rate of stearoyl-CoA desaturase mRNA degradation. The results presented in this study suggest that fibrates induce stearoyl-CoA desaturase activity and mRNA levels independently of peroxisome proliferation.

Molecular and functional characterization of LRP1 promoter polymorphism c.1-25 C>G (rs138854007).

The transcription of the Low-density lipoprotein receptor-related protein (LRP1) is upregulated by aggregated LDL (agLDL) and angiotensin II (AngII) in human vascular smooth muscle cells (hVSMC). The polymorphism c.1-25C>G creates a new GC-box in the LRP1 promoter recognized by Sp1/Sp3 transcription factors. The aims of this study were 1) to evaluate the impact of c.1-25C>G polymorphism on LRP1 transcriptional activity and expression, and 2) to examine the response of c.1-25C>G LRP1 promoter to LDL and AngII. EMSA and Luciferase assays in HeLa cells showed that -25G promoter has enhanced basal transcriptional activity and specific Sp1/Sp3 binding. hVSMC with GG genotype (GG-hVSMC) had higher LRP1 mRNA and protein levels, respectively than CC genotype (CC-hVSMC). EMSA assays showed that the polymorphism determines scarce amount of SRE-B/SREBP-2 complex formation and the failure of agLDL to further reduce these SRE-B/SREBP-2 complexes. Taken together, these results suggest that c.1-25C>G, by difficulting SREBP-2 binding, prevents SREBP-2 displacement required for LRP1 promoter response to LDL. In contrast, c.1-25C>G strongly favours Sp1/Sp3 binding and AngII-induced activity in Sp1/Sp3 dependent manner in GG-hVSMC. This increase is functionally translated into a higher capacity of GG-hVSMC to become foam cells from agLDL in presence of AngII. These results suggest that c.1-25C>G determines a lack of response to agLDL and an exacerbated response to AngII. It is thus conceivable that the presence of the polymorphism would be easily translated to vascular alterations in the presence of the pro-hypertensive autacoid, AngII.

PERMANENT GENETIC RESOURCES: Isolation and characterization of 12 microsatellite markers in the middle-spotted woodpecker (Dendrocopos medius).

Twelve polymorphic microsatellite loci were developed for Dendrocopos medius. Polymorphism was assessed for 27 individuals from the southwesternmost population of this woodpecker species. The number of alleles per locus ranged from three to seven, with observed heterozygosity values from 0.444 to 0.852. Genotypic frequencies conformed to Hardy-Weinberg equilibrium, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among habitat patches inhabited by D. medius. Three of the loci were polymorphic for Picoides articus.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity.

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and (1)H, (13)C, and (195)Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(mu-Cl)](2) . 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh(3) or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl(2)(DMSO(2)]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(mu-Cl)](2) has significant antiproliferative activity, although it is less active than cisplatin.

Differentially expressed genes between high-risk human papillomavirus types in human cervical cancer cells.

Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. Human papillomavirus (HPV) infection is the most important etiologic factor for CC. Of the oncogenic types, HPV16 and HPV18 are found in 60-70% of invasive CCs worldwide. HPV18 appears to be associated with a more aggressive form of cervical neoplasia than HPV16 infection. At present, there are no studies on differentially expressed cellular genes between transformed cells harboring HPV16 and HPV18 sequences. Based on previous complementary DNA microarray data from our group, 13 genes were found to be differentially overexpressed between HPV16- and HPV18-transformed cells. These genes were as follows: E6BP, UBE4A, C20orf14, ATF7, ABCC8, SLC6A12, WASF3, SUV39H1, SPAG8, CCNC, E2FFE, BIRC5, and DEDD. Differential expression of six selected genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). All real-time RT-PCRs confirmed differential expression between HPV18 and HPV(-) samples. The present work identifies genes from signaling pathways triggered by HPV transformation that could be differentially deregulated between HPV16(+) and HPV18(+) samples.