Saline spa water or combined water and UV-B for psoriasis vs conventional UV-B: lessons from the Salies de Béarn randomized study.

OBJECTIVE: To study the effects of UV-B therapy and saline spa water given alone or in combination for the treatment of psoriasis. DESIGN: Randomized, controlled, comparative study with blinded observers. SETTING: Salies de Béarn, saline spa water center located in the southwest of France. PARTICIPANTS: Seventy-one adult patients with psoriasis with a Psoriasis Area and Severity Index (PASI) score greater than 10. INTERVENTION: Patients were randomly assigned to 1 of 3 treatments: spa water alone (group A); UV-B 311-nm phototherapy alone (group B); and a combination of the 2 therapies (group C). The 3 groups were treated on a daily basis 5 days a week for a total of 21 days. MAIN OUTCOME MEASURES: Change in PASI score from baseline as determined by an investigator blinded to randomization; variation in quality of life, adverse effects, and long-term effects (1 year after treatment). RESULTS: Four patients dropped out because of secondary effects. Efficacy was similar in groups B and C, with changes in PASI of -64% and -55%, respectively at 3 weeks. For group A, change in PASI was -29%, thus showing a minor therapeutic effect of saline spa water alone and poor efficacy compared with groups B and C (P<.001). More adverse effects were reported in groups A and C but did not reach significance. Combined saline spa water and UV-B therapy had no sparing effect on UV-B dosages. One year after treatment, no long-term benefit could be attributed specifically to a given regimen, but the patients had overall significantly better PASI scores than at baseline. CONCLUSIONS: Saline spa water alone had a minor therapeutic effect in psoriasis, and the beneficial effect of bathing to enhance phototherapy was not demonstrated.

RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1.

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

Eukaryotic ribonucleases HI and HII generate characteristic hydrolytic patterns on DNA-RNA hybrids: further evidence that mitochondrial RNase H is an RNase HII.

RNase H activities from HeLa cells (either of cytoplasmic or mitochondrial origin), and from mitochondria of beef heart and Xenopus ovaries, have been tested with RNA-DNA substrates of defined length (20 bp) and sequence. Substrates were either blunt-ended, or presented DNA or RNA overhangs. The hydrolysis profiles obtained at early times of the digestion showed a good correlation between the class of RNase H, either type I or II assigned according to biochemical parameters, whatever the organism. Consequently, the pattern of primary cuts can be considered as a signature of the predominant RNase H activity. For a given sequence, hydrolysis profiles obtained are similar, if not identical, for either blunt-ended substrates or those presenting overhangs. However, profiles showed variations depending on the sequence used. Of the three sequences tested, one appears very discriminatory, class I RNases H generating a unique primary cut 3 nt from the 3' end of the RNA strand, whereas class II RNases H generated two simultaneous primary cuts at 6 and at 8 nt from the 5' end of the RNA strand. Hydrolysis profiles further confirm the assignation of the mitochondrial RNase H activity from HeLa cells, beef heart and Xenopus oocytes to the class II.

Detection of proteins binding to short RNA.DNA hybrids or short antisense oligonucleotides in Xenopus laevis oocytes and human macrophage cell extracts by photoaffinity radiolabeling.

Using a 12 base pair RNA.DNA hybrid, substituted with bromouracil on either the RNA or DNA strand, we have detected by photoaffinity radiolabeling a limited set of proteins able to bind to RNA.DNA hybrids in both Xenopus oocyte extracts and human macrophage extracts. Resulting patterns of crosslinked proteins were highly dependent on the strand (DNA or RNA) that was substituted. With one exception, none of the proteins investigated in competition experiments was found to be absolutely specific for RNA.DNA hybrids, as at least one other nucleic acid, either single-stranded DNA or single-stranded RNA, was found to compete efficiently. None of the proteins detected in this assay correspond to the size expected for RNases H. Using the same methodology, we have detected proteins that bind to short oligodeoxyribonucleotides. Although we have essentially detected in Xenopus oocytes one prominent protein of approximately 75 kDa, corresponding to replication protein A (RPA) whatever the oligonucleotide used, the patterns obtained with extracts of human macrophages were more complex and dependent on the oligonucleotide used. If a protein corresponding to RPA was observed most of the time, other crosslinks of similar or sometimes higher intensity were also detected. Interestingly, among these, one protein of 35 kDa appears paradoxically to bind and crosslink to a dodecamer but not to an octadecamer containing the same sequence placed either at its 3'-end or 5'-end.

[Use of transjugular intrahepatic portosystemic shunt (TIPS) in an 84 year-old patient during acute variceal bleeding]. / Utilizacion del shunt portosistemico intrahepatico transyugular (TIPS) en un paciente 84 años durante un episodio de hemorragia aguda por varices esofagicas.

The transjugular intrahepatic protosystemic shunt (TIPS) is a nonsurgical method with low mortality which does not interfere with subsequent liver transplantation. We describe the case of an old male patient with chronic hepatopathy who was admitted because of an episode of variceal bleeding. Due to the failure of the medical treatment and sclerotherapy, coronarian stomachic vein was embolized with a coil and a TIPS was performed during the acute episode of variceal bleeding. He evolved favorably and after a year of the procedure remains asymptomatic.

Rupture of mitral chorda tendinea following blunt chest trauma.

Acute mitral insufficiency, originated from rupture of mitral chordae tendineae secondary to nonpenetrating thoracic trauma, is an unusual condition. This diagnosis is difficult to establish because physical examination, electrocardiogram, and cardiac enzymes are neither sensitive nor specific. The diagnosis of rupture must be quickly established because this disorder may be fatal. This paper reports the case of a patient with acute mitral insufficiency secondary to a mitral valve chord rupture a week after a nonpenetrating thoracic trauma.

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

Purification and characterization of human ribonuclease HII.

A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.

Characterization and subcellular localization of ribonuclease H activities from Xenopus laevis oocytes.

Ribonuclease H activities present in fully grown Xenopus oocytes were investigated by using either liquid assays or renaturation gel assays. Whereas the test in solution detected an apparently unique class I ribonuclease H activity, the activity gels did not detect this enzyme but another one with the molecular weight expected for a class II ribonuclease H. The ribonuclease HI was found to be primarily concentrated in the germinal vesicle, but around 5% of this activity was detectged in the cytoplasm and may correspond to the activity involved in antisense oligonucleotide-mediated destruction of messenger RNAs. The concentration of this class I ribonuclease H in oocytes is similar to that in somatic cells. The class II ribonuclease H remained undetectable by the test in solution because its activity was cryptic. On activity gel, a polypeptide with the apparent molecular mass of 32 kDa, expected for a ribonuclease HII, was found to be concentrated in mitochondria although no RNase H activity could be detected by using the liquid assay. Based on sedimentation studies, we hypothesize that the apparent absence of RNase H activity in solution could be the result of the association of this 32-kDa polypeptide with other polypeptides, or possibly nucleic acids, to form a multimer of, until now, unknown function.

Production of double-stranded RNA during synthesis of bromouracil-substituted RNA by transcription with T7 RNA polymerase.

Using T7 RNA polymerase we synthesized a short oligoribonucleotide containing bromouracil by in vitro transcription of a synthetic DNA template. Whereas the major transcript obtained had the expected size and was apparently homogeneous on a sequencing gel, additional analysis revealed the presence of double-stranded RNA in this preparation. As this was not observed when the same template was transcribed in the presence of uracil, we hypothesize that bromouracil promoted the apparition of double-stranded 'parasitic' RNA presumably by favouring priming for the RNA-dependent RNA synthesis of the T7 RNA polymerase or by facilitating an end-to-end copy mechanism.

RNA template-directed RNA synthesis by T7 RNA polymerase.

In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage T7 RNA polymerase, a major transcript was produced that was much longer than expected. Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template. In reactions containing only an RNA template, the rate of incorporation of NTPs was quite comparable to DNA-dependent transcription. RNA template-directed RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multistep extension reaction converting the oligonucleotide into a final product longer than twice its original length. Presumably, such a process could have generated some of the RNAs found to be efficiently replicated by T7 RNA polymerase.

Sensitive detection of low levels of ribonuclease H activity by an improved renaturation gel assay.

Renaturation gel assays are good tools to assign enzymatic activities to protein bands. First, proteins are separated by denaturating electrophoresis on substrate-containing gels. Then, following the elimination of the denaturing agent, polypeptides are allowed to renature, thus leading to the degradation of the embedded substrate at positions at which the corresponding activity has moved. Nevertheless, this in situ technique does not only reflect a certain amount of enzyme activity, it also depends upon the ability of an enzyme to renature. Here we present a renaturation gel assay procedure with an improved sensitivity and discuss the detection of E. coli and human ribonuclease H activities as an example.

Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate.

Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is cleaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions for the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates. In these conditions, however, the remaining activity found in the wheat germ extract was three times higher than the activity found in the rabbit reticulocyte lysate. Whether this difference can by itself explain the indicated differences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H.

[Antisense oligonucleotides: tools of molecular genetics and therapeutic agents]. / Les oligonucléotides antisens: outils de génétique moléculaire et agents thérapeutiques.

The binding of an oligodeoxynucleotide, so-called anti-sense, to the complementary sequence of a messenger RNA can prevent the synthesis of the encoded protein. This approach constitutes a very efficient and specific means to artificially regulate gene expression. Numerous chemical modifications have been introduced into synthetic oligos in order to provide them with properties that unmodified molecules do not display. For instance, oligos built up with methylphosphonate, phosphorothioate and alpha-anomer units lead to molecules that are resistant to DNases. Acridine-linked oligos exhibit an increased affinity for the target sequence due to the intercalation of the dye into the oligo/RNA duplex. Two different mechanisms account for translation inhibition by antisense oligos. Inhibition of the elongation step results only from the induced cleavage of the target RNA by RNase-H. In contrast, oligos targeted upstream of the AUG initiation codon can block the initiation step through an RNase-H independent mechanism. As a consequence, methylphosphonate- and alpha-oligos, which do not elicit RNase-H activity, targeted to the 5' region, are efficient antisense; but they are inactive if targeted to the coding sequence. Experiments performed with antisense oligos in cell-free extracts supported the notion that the mini-exon sequence, acquired by trans-splicing, was present on every message in trypanosomatids and on some of them in nematodes. Furthermore, an acridine-linked oligo complementary to the mini-exon sequence of Trypanosoma brucei induced a lethal effect on cultured procyclics. Therefore these compounds constitute promising tools in molecular genetics and could open new routes to rationally tailor therapeutic agents.

5 fluorouracilo en la cirugía filtrante del glaucoma / 5 fluoruoracil in filtering surgery of the glaucoma

Los citostáticos aplicados en forma tópica constituyen una nueva línea de investigación en la cirugía filtrante externa del glaucoma, cuya aplicación en clínica comienza a evaluarse. Tienen como objetivo lograr un manejo adecuado de la cicatrización epiescleral y conjuntival que conduzca a la obtención de una vesícula de filtración permanente que controle la presión ocular en niveles normales. En esta investigación se usa el 5 fluorouracilo en inyecciones subconjuntivales a partir de las 24 horas del postoperatorio en 28 ojos portadores de glaucoma de mal pronóstico quirúrgico

Phosphoroselenoate oligodeoxynucleotides: synthesis, physico-chemical characterization, anti-sense inhibitory properties and anti-HIV activity.

Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.

Spinal subarachnoid hematoma complicating lumbar puncture: diagnosis and management.

Two patients with altered hemostatic mechanisms developed spinal subarachnoid hemorrhage after difficult lumbar punctures. One patient had received anticoagulation therapy soon after lumbar puncture and the other had a low platelet count (63,000/mm3) at the time of lumbar puncture. In both patients a hematoma evolved, producing blockage of cerebrospinal fluid flow. Clinical manifestations consisted of severe back and radicular pain with sphincteric disturbances followed by rapidly developing severe paraparesis. Of the methods of radiographic evaluation that were used, including computed tomography (CT) without contrast enhancement, myelography, CT with intrathecally administered contrast medium, and magnetic resonance imaging, we found the best study to be myelography via lateral cervical puncture followed by CT. Unfortunately, diagnosis was delayed, and surgical evacuation of the hematomas did not substantially improve the patients' conditions. The salient clinical and radiographic features of this disorder and its pathophysiology are reviewed. Prompt recognition of these lesions is necessary so that surgical intervention may maximize chances of recovery.