Clinical implementation of a multiplex PCR detection method for HPV causing plantar warts and genotype distribution in different geographical areas of Spain.

Human papillomavirus is the ethological agent of various tumors, including plantar warts as one of the most frequent clinical presentations. Diagnosis of these warts continues to be mainly clinical, and a significant incidence of misdiagnosis leads to inadequate treatment. The aim of this study is to implement and validate a multiplex PCR detection method in the clinical setting to detect HPV in samples and to study genotype distribution in Spain to improve future molecular diagnostics. Viral DNA was extracted from 128 samples of clinically suspected plantar warts from various locations in Spain. A multiplex PCR was run alongside internal controls, and amplicons were processed for sequencing and HPV genotyping. The method was validated by assessing both inter- and intra-run repeatability. The PCR detection method returned 81.2 % (n = 104) positive results in the samples tested. Inter- and intra-run repeatability tests showed excellent intra-run agreement (κ = 1.00, p < 0.001) and good inter-run agreement (κ = 0.737, p < 0.001). The most frequent HPV type was HPV1, followed by HPV27, showing a statistical difference between the distribution of HPV genotypes in different areas of Spain. Clinical implementation of a DNA PCR detection method for plantar warts can avoid 18.8 % of unnecessary treatments in doubtful cases, and the method is reliable and validated for the purpose. HPV types show an asymmetric geographical distribution that should be considered for diagnosis and treatment.

minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila.

The development of the adult central nervous system of Drosophila requires a precise and reproducible pattern of neuroblast proliferation during postembryonic neurogenesis. We show here that mutations in the minibrain (mnb) gene cause an abnormal spacing of neuroblasts in the outer proliferation center (opc) of larval brain, with the implication that mnb opc neuroblasts produce less neuronal progeny than do wild type. As a consequence, the adult mnb brain exhibits a specific and marked size reduction of the optic lobes and central brain hemispheres. The insufficient number of distinct neurons in mnb brains is correlated with specific abnormalities in visual and olfactory behavior. The mnb gene encodes a novel, cell type-specific serine-threonine protein kinase family that is expressed and required in distinct neuroblast proliferation centers during postembryonic neurogenesis. The mnb kinases share extensive sequence similarities with kinases involved in the regulation of cell division.

The Down syndrome candidate dual-specificity tyrosine phosphorylation-regulated kinase 1A phosphorylates the neurodegeneration-related septin 4.

The dual-specific kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) is the mammalian orthologue of the Drosophila minibrain (MNB) protein kinase and executes diverse roles in neuronal development and adult brain physiology. DYRK1A is overexpressed in Down syndrome (DS) and has recently been implicated in several neurodegenerative diseases. In an attempt to elucidate the molecular basis of its involvement in cognitive and neurodegeneration processes, we searched for novel proteins interacting with the kinase domain of DYRK1A in the adult mouse brain and identified septin 4 (SEPT4, also known as Pnutl2/CDCrel-2). SEPT4 is a member of the group III septin family of guanosine triphosphate hydrolases (GTPases), which has previously been found in neurofibrillary tangles of Alzheimer disease brains and in alpha-synuclein-positive cytoplasmic inclusions in Parkinson disease brains. In transfected mammalian cells, DYRK1A specifically interacts with and phosphorylates SEPT4. Phosphorylation of SEPT4 by DYRK1A was inhibited by harmine, which has recently been identified as the most specific inhibitor of DYRK1A. In support of a physiological relation in the brain, we found that Dyrk1A and Sept4 are co-expressed and co-localized in neocortical neurons. These findings suggest that SEPT4 is a substrate of DYRK1A kinase and thus provide a possible link for the involvement of DYRK1A in neurodegenerative processes and in DS neuropathologies.

Segregation of postembryonic neuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval brain.

Due to its intermediate complexity and its sophisticated genetic tools, the larval brain of Drosophila is a useful experimental system to study the mechanisms that control the generation of cell diversity in the CNS. In order to gain insight into the neuronal and glial lineage specificity of neural progenitor cells during postembryonic brain development, we have carried an extensive mosaic analysis throughout larval brain development. In contrast to embryonic CNS development, we have found that most postembryonic neurons and glial cells of the optic lobe and central brain originate from segregated progenitors. Our analysis also provides relevant information about the origin and proliferation patterns of several postembryonic lineages such as the superficial glia and the medial-anterior Medulla neuropile glia. Additionally, we have studied the spatio-temporal relationship between gcm expression and gliogenesis. We found that gcm expression is restricted to the post-mitotic cells of a few neuronal and glial lineages and it is mostly absent from postembryonic progenitors. Thus, in contrast to its major gliogenic role in the embryo, the function of gcm during postembryonic brain development seems to have evolved to the specification and differentiation of certain neuronal and glial lineages.

Protein S4 is near the elongation factor G binding site in the ribosome.

Using a mild iodination method for protein radioactive labeling, it has been shown that elongation factor G, when bound to the ribosome as EFG-GDP-fusidic acid complex, protects protein S4 from labeling. The results indicate that protein S4 is probably near the ribosomal EFG binding site.

Synthesis and biological activity of photoactive derivatives of erythromycin.

Five photoactive derivatives of erythromycin have been synthesized by linking to 9(S)-aminoerythromycin either an aryl azide or a p-nitrophenyl ether. One derivative is an amide formed by reaction with (5-azido-2-formyl-phenoxy)acetic acid. Three derivatives are also amides, synthesized with 4-(p-nitroguaiacoxy)butanoic acid as a photoreactive group either directly or by interposing an amino acid (glycine or tyrosine). The last derivative is the product of the aldehyde condensation of aminoerythromycin with 10-(p-nitroguaiacoxy)decanal. Two of these derivatives can easily be made radioactive for affinity labeling studies either by reduction with [3H]borohydride (aryl azide derivative) or by 125I iodination (4-(p-nitroguaiacoxy)tyrosyl derivative). Although affected to different extents, the five erythromycin derivatives are biologically active and bind to the erythromycin-specific site on the bacterial ribosomes. In addition, the introduction of these groups changes the erythromycin inhibition pattern of peptide bond model reactions.

A method for pulse and chase BrdU-labeling of early chick embryos.

Pulse and chase BrdU labeling of early chick embryos represents a serious technical problem due to the hindrance of removing unincorporated BrdU after the pulse. We have developed a simple method that allows BrdU washout and control of pulse/chase duration. In this method, BrdU pulses are carried out in ovo. Afterwards, embryos are removed from the yolk, BrdU is washed out, and the embryos are maintained in a wholemount culture. Under these conditions, HH8-12 embryos continue with their normal development for at least 30 h. Morphological development of the nervous system and cell cycle kinetics of precursor cells seem to be normally maintained in cultured embryos. To prove the feasibility of the method, it has been applied to determine the onset of TUJ1 expression. TUJ1 is frequently considered an early neuronal marker, yet some reports have shown its expression in dividing progenitor cells and differentiating neurons. The application of this new method demonstrates that TUJ1 is expressed in newborn neurons as early as 1 h after cell cycle exit.

The MNB/DYRK1A protein kinase: neurobiological functions and Down syndrome implications.

Major attention is being paid in recent years to the genes harbored within the so called Down syndrome Critical Region of human chromosome 21. Among them, those genes with a possible brain function are becoming the focus of intense research due to the numerous neurobiological alterations and cognitive deficits that Down syndrome individuals have. MNB/DYRK1A is one of these genes. It encodes a protein kinase with unique genetic and biochemical properties, which have been evolutionarily conserved from insects to humans. MNB/DYRK1A is expressed in the developing brain where it seems to play a role in proliferation of neural progenitor cells, neurogenesis, and neuronal differentiation. Although at a lower level, MNB/DYRK1A is also expressed in the adult brain where, as judged by the phenotype of mutant and transgenic animals, it may be involved in learning and memory. Nevertheless, most of the molecular mechanisms underlying these functions remain to be unraveled. In this review we compile and discuss experimental evidences, which support the involvement of MNB/DYRK1A in several neuropathologies and cognitive deficits of Down syndrome.

The MNB/DYRK1A protein kinase: genetic and biochemical properties.

The "Down syndrome critical region" of human chromosome 21 has been defined based on the analysis of rare cases of partial trisomy 21. Evidence is accumulating that DYRK1A, one of the 20 genes located in this region, is an important candidate gene involved in the neurobiological alterations of Down syndrome. Both the structure of the DYRK1A gene and the sequence of the encoded protein kinase are highly conserved in evolution. The protein contains a unique assembly of structural motifs outside the catalytic domain, including a nuclear localization signal, a PEST region, and a repeat of 13 consecutive histidines. MNB/DYRK1A and related kinases are unique among serine/threonine-specific protein kinases in that their activity depends on tyrosine autophosphorylation in the catalytic domain. Also, evidence is accumulating that mRNA levels of MNB/DYRK1A are subject to tight regulation. A number of putative substrates of MNB/DYRK1A have emerged in the recent years, the majority of them being transcription factors. Although the function of MNB/DYRK1A in intracellular signalling and regulation of cell function is still poorly defined, current evidence suggests that the kinase may play a role in the regulation of gene expression.

Site of covalent attachment of alpha-scorpion toxin derivatives in domain I of the sodium channel alpha subunit.

Purified and reconstituted sodium channels from rat brain have been photoaffinity labeled with a photoactivable derivative of the alpha-scorpion toxin V from Leiurus quinquestriatus (LqTx). A battery of sequence-specific antibodies has been used to determine which of the peptides produced by chemical and enzymatic cleavage of the photolabeled sodium-channel alpha subunit contain covalently attached LqTx. Nearly all of the covalently attached LqTx is found within homologous domain I. Two site-directed antisera, which recognize residues 317 to 335 and residues 382 to 400, respectively, specifically immunoprecipitate a 14-kDa peptide produced by CNBr digestion to which LqTx is covalently attached. It is proposed that a portion of the receptor site for alpha-scorpion toxins is formed by peptide segment(s) between amino acid residues 335 and 378 which is located in an extracellular loop between transmembrane helices S5 and S6 of homologous domain I of the sodium channel alpha subunit.

Components of the macrolide binding site on the ribosome.

Carbomycin A, niddamycin and tylosin, macrolide antibiotics that inhibit ribosomal activity, have alpha-beta unsaturated ketone groups in their structure that make them photoreactive. These drugs can also take part in thermic reactions, probably through an addition mechanism to the double bond. Given of the photoactivity and thermic reactivity of their molecules, affinity labeling of the macrolide binding site on the ribosome has been performed using radioactive derivatives of these drugs. After either irradiating or incubating samples containing antibiotics and ribosomal particles, radioactivity appears covalent associated to the proteins. Ribosomal protein L27 is the major labeled component, indicating that this polypeptide, which seems to be part of the peptidyl transferase centre of the ribosome, also plays an important role on the macrolide binding site.

Ribosome structure: binding site of macrolides studied by photoaffinity labeling.

The macrolide antibiotics carbomycin A, niddamycin, and tylosin have been radioactively labeled by reducing their aldehyde group at the C-18 position. Dihydro derivatives with specific activities around 2.5 Ci/mmol can be obtained that, although partially affected in their activity, still bind to the ribosomes with high affinity. The presence in the chemical structure of these antibiotics of alpha-beta-unsaturated ketone groups makes them photochemically reactive, and by irradiation above 300 nm, covalent incorporation of the radioactive dihydro derivatives into ribosomes has been achieved. The covalent binding seems to take place at the specific binding sites for macrolides as deduced from binding saturation studies and competition experiments with unmodified drugs. Analysis of the ribosomal components labeled by the drugs indicated that most radioactivity is associated with the proteins L27, L2, and L28 when 50S subunits are labeled, and with L27, L2, L32/33, S9, and S12 in the case of 70S ribosomes. These results agree well with a model of macrolides' mode of action that assumes an interaction of the drug at the peptidyl transferase P site that would block the exit channel for the growing peptide chain.

Photoaffinity labeling of the receptor site for alpha-scorpion toxins on purified and reconstituted sodium channels by a new toxin derivative.

1. A methyl-4-azidobenzimidyl (MAB) derivative of the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) specifically labels only the alpha subunit of the rat brain sodium channel in synaptosomes or in purified and reconstituted sodium-channel preparations. 2. Unlike previous photoreactive toxin derivaties, binding and photolabeling by MAB-LqTx are allosterically modulated by tetrodotoxin and batrachotoxin, as observed for native LqTx binding to sodium channels in synaptosomes. 3. Proteolytic cleavage of the alpha subunit photolabeled with MAB-LqTx shows that the label is located within a 60 to 70-kDa protease-resistant core structure in domain I of the sodium-channel alpha subunit. 4. MAB-LqTx will be valuable in further defining the structure-activity relationships at the alpha-scorpion toxin receptor site.

Iodination of p-nitrophenyl ethers used as photolabels.

A system for radioactive labeling of compounds of biological interest that, due to their low electronic density, cannot be labeled by the standard iodination techniques is described. Using p-nitroanisole as a model, we have prepared 2-[125I]iodo-4-nitroanisole by treatment with thallium trifluoroacetate, with later displacement of the thallium by iodide according to A. McKillop et al. (J. Amer. Chem. Soc. 93, 4841-4844 (1970)). The labeled iodonitroanisole has been used as a photoactive reagent to label a protein (bovine serum albumin), showing that under the irradiation conditions used, the label is incorporated into the polypeptide mainly through modification of epsilon-amino groups of the lysine residues.

Immunochemical characterization and developmental expression of Shaker potassium channels from the nervous system of Drosophila.

We have raised antisera against recombinant peptides expressed from cDNAs fragments common to all splicing variants generated at the Shaker locus of Drosophila and used them as a tool to biochemically characterize these channel proteins. This antisera succeeded in detecting the expression of multiple Shaker potassium channels (Sh Kch), proteins with variable molecular mass (65-85 kDa) and pI (5.5-7). Additionally, for first time, specific Sh proteins of 40-45 kDa most probably corresponding to some of the so-called short Sh cDNAs previously isolated by others have been identified. Using genetic criteria, it has been determined that at least a good part of this variety of proteins is generated by alternative splicing. Developmental experiments show a double wave of Sh Kch channel expression with a first pick at the third instar larvae stage, a minimum at the beginning of puparation, and the highest plateau 36 h after hatching of adult flies. The pattern of Sh splice variants changes dramatically throughout development. A detergent-resistant fraction with about 50% of Sh Kch which seems to be anchored to submembranous structures has been found. Finally, other biochemical properties of Sh Kch, like membrane fractionation and glycosylation, are also described.

Reaction of some macrolide antibiotics with the ribosome. Labeling of the binding site components.

Radioactive carbomycin A, niddamycin, tylosin, and spiramycin, but not erythromycin, can be covalently bound to Escherichia coli ribosomes by incubation at 37 degrees C. The incorporation of radioactivity into the particles is inhibited by SH- and activated double bond containing compounds but not by amino groups, suggesting that the reactions may take place by addition to the double bond present in the reactive antibiotics. This thermic reaction must be different from the photoreaction described for some of these macrolides [Tejedor, F., & Ballesta, J. P. G. (1985) Biochemistry 24, 467-472] since tylosin, which is not photoincorporated, is thermically bound to ribosomes. Most of the radioactivity is incorporated into the ribosomal proteins. Two-dimensional gel electrophoresis of proteins labeled by carbomycin A, niddamycin, and tylosin indicates that about 40% of the radioactivity is bound to protein L27; the rest is distributed among several other proteins such as L8, L2, and S12, to differing extents depending on the drug used. These results indicate, in accordance with previous data, that protein L27 plays an important role in the macrolide binding site, confirming that these drugs bind near the peptidyl transferase center of the ribosome.

Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding.

Incubation of purified rat brain sodium channels at 37 degrees C or at high ionic strength causes a concomitant loss of saxitoxin-binding activity and dissociation of beta 1 subunits. Reaction with hydrophilic carbodiimides produced a resistance against the loss of saxitoxin binding and caused covalent cross-linking of alpha, beta 1, and beta 2 subunits. In the presence of saxitoxin, this cross-linking reaction led to formation of a state with increased affinity for saxitoxin. However, analysis of the concentration dependence of covalent cross-linking and its inhibition by hydrophilic nucleophiles showed that the stabilization of the saxitoxin-binding activity was due to the formation of a small number of isopeptide bonds in the alpha subunit rather than to cross-linking of alpha and beta 1 subunits. In the presence of amine nucleophiles, carbodiimides caused loss of saxitoxin binding, which was prevented in the presence of the toxin. Nucleophiles yielding positively charged amide products were more effective than those forming uncharged or negatively charged products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availability of carboxyl groups for reaction was increased, providing evidence for a toxin-induced conformational change on binding. These results are considered in terms of an allosteric model of saxitoxin binding, in which the functional form of the sodium channel having high affinity for saxitoxin can be stabilized against inactivation by noncovalent interactions with beta 1 subunits, binding of saxitoxin and tetrodotoxin, or intramolecular cross-linking of amino acid residues within the alpha subunit.

Diverse expression and distribution of Shaker potassium channels during the development of the Drosophila nervous system.

The spatio-temporal expression of Shaker (Sh) potassium channels (Kch) in the developing and adult nervous system of Drosophila has been studied at the molecular and histological level using specific antisera. Sh Kch are distributed in most regions of the nervous system, but their expression is restricted to only certain populations of cells. Sh Kch have been found in the following three locations: in synaptic areas of neuropile, in axonal fiber tracks, and in a small number of neuronal cell bodies. This wide subcellular localization, together with a diverse distribution, implicates Sh Kch in multiple neuronal functions. Experiments performed with Sh mutants that specifically eliminate a few of the Sh Kch splice variants clearly demonstrate an abundant differential expression and usage of the wide repertoire of Sh isoforms, but they do not support the idea of extensive segregation of these isoforms among different populations of neurons. Sh Kch are predominantly expressed at late stages of postembryonic development and adulthood. Strikingly, wide changes in the repertoire of Sh splice isoforms occur some time after the architecture of the nervous system is complete, indicating that the expression of Sh Kch contributes to the final refinements of neuronal differentiation. These late changes in the expression and distribution of Sh Kch seem to correlate with activity patterns suggesting that Sh Kch may be involved in adaptative mechanisms of excitability.

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes.

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.