Search

Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes scavenging biogenic or exogenous amines and generating hydrogen peroxide. We asked whether human adipose tissue could express SSAO. Since hydrogen peroxide exhibits pharmacological insulin-like effects, we also tested whether its endogenous production by SSAO could mimic several insulin effects on adipocytes, such as stimulation of glucose uptake and inhibition of lipolysis. The benzylamine oxidation by human adipose tissue was inhibited by semicarbazide or hydralazine and resistant to pargyline or selegiline. It was due to an SSAO activity localized in adipocyte membranes. A protein of 100-kDa and a 4-kb mRNA corresponding to SSAO were identified in either mammary or abdominal subcutaneous fat depots. In isolated adipocytes, SSAO oxidized similarly benzylamine and methylamine that dose dependently stimulated glucose transport in a semicarbazide-sensitive manner. Antioxidants also inhibited the benzylamine and methylamine effects. Moreover, the ability of diverse substrates to be oxidized by adipocytes was correlated to their effect on glucose transport. Benzylamine and methylamine exerted antilipolytic effects with a maximum attained at 1 mM. These results show that human adipocytes express a membrane-bound SSAO that not only readily oxidizes exogenous amines and generates H(2)O(2), but that also interplays with glucose and lipid metabolism by exerting insulin-like actions. Based on these results and the fact that variations in plasma levels of the soluble form of SSAO have been previously reported in diabetes, we propose that determination of adipocyte SSAO, feasible on subcutaneous microbiopsies, could bring relevant information in pathologies such as obesity or diabetes.

Objectives: To determine whether the peripersonal and intrapersonal buccal space can be affected by a hemispheric stroke and to evaluate the clinical signs resulting from buccal neglect., Methods: A prospective study comparing 2 groups of patients with hemiplegia, 1 with a right hemispheric lesion and the other with a left hemispheric lesion. Patients were selected consecutively on the basis of specific criteria at least 1 month after stroke., Results: Buccal hemineglect was usually concomitant with other hemineglect phenomena resulting from lesions of the right hemisphere (10 of 12 in right lesions and 1 of 12 in left lesions). Clinical signs associated with this condition consisted of impaired swallowing (retention, defective insalivation, presence of food debris in the left hemibuccal space, loss of saliva from the left side of the mouth, and choking); loss of the ability to perceive salty, sweet, or acid tastes; and impaired buccal representation. These problems were usually incorrectly diagnosed initially. Outcome was usually favorable, but functional disorders persisted in some patients for more than 18 months. The underlying attention and representation mechanisms are discussed with reference to experimental lesions of the postarcuate (area 6) cortex in rhesus monkeys. The area around the mouth may be considered to be, as in monkeys, a peripersonal space, ie, probably of little functional importance. The lesion may involve area 6 or its projections to the thalamus or posterior parietal cortex., Conclusions: Buccal hemineglect, which is likely to cause social embarrassment, should be considered whenever the oral phase of swallowing is impaired in a context of neglect syndromes. Prophylactic measures and rehabilitation can reduce the impact and complications of the condition (food bolus).

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

An in vitro assay based on the expression of Fluci reporter gene under the translational control of HCV IRES was used to evaluate and screen compound libraries. A structure-activity relationship study on a phenazine hit was conducted. Our data suggest that an intact phenazine or phenazine-like core with two distal polar substitutions is crucial for potency.

Neuropeptide Y (NPY) increases food intake through the action of hypothalamic NPY receptors. At least six subtypes of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) receptors have been identified in mice. Although the involvement of Y1 and Y5 receptors in feeding regulation has been suggested, the relative importance of each of these NPY receptors and the participation of a novel feeding receptor are still unclear. To address this issue, we generated a Y1 receptor-deficient (Y1-/-) and a Y5 receptor-deficient (Y5-/-) mouse line in which we directly compared the orexigenic effects of NPY and its analogs after intracerebroventricular (icv) administration. The icv NPY-induced food intake was remarkably reduced in Y1-/- mice, but was not significantly altered by inactivation of the Y5 receptor. The Y1 receptor therefore plays a dominant role in NPY-induced feeding. Stimulation of feeding by moderately selective Y5 agonists [PYY-(3-36), human PP, and bovine PP] was reduced in Y5-/- mice, although food intake did not decrease to vehicle control levels. These results indicate that the Y5 receptor functions as one of the feeding receptors. In addition, the finding that Y5-preferring agonists still induce food intake in Y5-/- mice suggests a role for another NPY receptor(s), including the possibility of novel NPY receptors. Surprisingly, despite the limited efficacy of PYY-(3-36) and PPs at the Y1 receptor, food consumption induced by these agonists was significantly diminished in Y1-/- mice compared with that in wild-type controls. These observations suggest that the feeding stimulation induced by NPY and its analogs may be directly or indirectly modulated by the action of the Y1 receptor. We conclude that multiple NPY receptors, possibly including the novel feeding receptor, are involved in the feeding response evoked by NPY and its analogs. Among them, the Y1 receptor plays a key role in NPY-induced feeding in mice.

Octopamine is known to exert adrenergic effects in mammals although specific octopamine receptors have been cloned only in invertebrates. It has been shown that octopamine can stimulate alpha(2)-adrenoceptors (ARs) in Chinese hamster ovary cells transfected with human alpha(2)-ARs. More recently, we reported that octopamine stimulates lipolysis through beta(3)-rather than beta(1)-or beta(2)-AR activation in white adipocytes from different mammalian species. The present study was thus undertaken to further characterize the adrenergic properties of octopamine. For this purpose, several biological processes known to be regulated by adrenergic stimulation were studied in response to octopamine, noradrenaline, adrenaline and tyramine in white adipocytes from different mammals. First, octopamine was fully lipolytic in garden dormouse and Siberian hamster while tyramine was ineffective. Although being around one hundred-fold less potent that noradrenaline, octopamine was slightly more potent in these hibernators known for their high sensitivity to beta(3)-AR agonists than in rat and chiefly more active than in human adipocytes known for their limited responses to beta(3)-AR agonists. Second, octopamine reduced insulin-dependent glucose transport in rat fat cells, a response also observed with noradrenaline and selective beta(3)-AR agonists but not with beta(1)-or beta(2)-agonists. Third, human adipocytes, which endogenously express a high level of alpha(2)-ARs, exhibited a clear alpha(2)-adrenergic antilipolytic response to adrenaline but not to octopamine. Moreover, octopamine exhibited only a very weak affinity for the alpha(2A)-ARs labeled by [3H]RX821002 in human adipocyte membranes. In Syrian hamster adipocytes, which also possess alpha(2)-ARs, octopamine induced only a weak antilipolysis. Finally, octopamine was a substrate of fat cell amine oxidases, with an apparent affinity similar to that of noradrenaline. All these results demonstrate that octopamine, tyramine noradrenaline and adrenaline can be degraded by adipocyte amine oxidases. However these biogenic amines interact differently with adipocyte adrenoceptors: tyramine is inactive, adrenaline and noradrenaline activate both beta- and alpha(2)-ARs while octopamine activates only beta(3)-ARs and is devoid of alpha(2)-adrenergic agonism. Thus, octopamine could be considered as an endogenous selective beta(3)-AR agonist.

The efficacy of intravitreal foscarnet injections was evaluated in a rabbit model of Herpes simplex virus type-1 (HSV-1) retinitis. In untreated infected animals, viral titration revealed that the optic chiasm, vitreous and chorioretina were positive for HSV-1. On the other hand, foscarnet treatment significantly decreased the viral count in the chorioretina when compared to the untreated group. Immunolocalization of HSV in untreated infected animals clearly showed infected cells in the outer and inner layers of the retina and also in the ciliary body of the eye. Clinical examination by indirect ophthalmoscopy indicated an absence of optic nerve congestion and a lower level of vitritis in foscarnet treated animals compared to the untreated group. It is concluded that intravitreal injections of foscarnet reduced the viral titer in the chorioretina in a rabbit model of HSV-1 retinitis. This route of administration might be valuable for the treatment of CMV retinitis in AIDS patients with sight threatening lesions or intolerance to intravenous anti-CMV drugs.

Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time.

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable., (Copyright 1998 Academic Press)

It has been shown previously that protein kinase A (PKA) maintains Xenopus oocytes arrested at G2, at least in part by preventing c-mos translation, but how PKA controls c-mos translation is not known. Using microinjection of recombinant c-mos, which still activates MAP kinase in the presence of active PKA, we have found that PKA does not exert any effect on translation of endogenous c-mos if MAP kinase is first activated. Even though they accumulate c-mos and contain MAP kinase activity as high as control oocytes, oocytes do not exit G2 in the presence of active PKA. These results are discussed in connection with recent findings on regulation of c-raf activity.

In a chromatographic system using human serum albumin (HSA) as a stationary phase, D,L dansyl amino acids as solutes, and sucrose as a mobile-phase modifier, a study on the surface tension effect of sugar on compound retention was carried out by varying the salting-out agent concentration c and the column temperature T. The thermodynamic parameters for solute transfer from the mobile to the stationary phase were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction between solute and HSA was independent of the molecular structure of the dansyl amino acids and the mobile-phase composition. An analysis of the experimental variations in the retention factor and the enantioselectivity values with c was performed using a theoretical model. It was shown that the decrease in solute retention accompanying the sucrose concentration increase was principally governed by a structural rearrangement of the binding cavity due to the increased surface tension effects. The cavity apolar residues were assumed to fold out of contact with the medium in order to reduce the surface area accessible to sucrose molecules, thus implying a restriction of the curvature radius of the cavity. Such behavior caused a decrease in the hydrophobic interaction for ligand binding on HSA explaining the observed thermodynamic parameter trends over the sucrose concentration range.

The high-performance liquid chromatography retention mechanism of a series of six imidazole derivatives was investigated over a wide range of mobile-phase compositions, pH, and column temperatures using a beta-cyclodextrin (beta-CD)-bonded chiral stationary phase. Thermodynamic constants for the transfer of a solute from the mobile to the beta-CD stationary phase were determined. Different van't Hoff plot shapes were observed with mobile-phase pH values, indicating a change in the retention mechanism. Enthalpy-entropy compensation revealed that the solute retention mechanism was independent of the compound molecular structure, the same at pH 7 and 7.5, but changed at pH 6.5, 8, and 8.5. Differential scanning calorimetry and thermogravimetric analysis were used to show different thermal features for the beta-CD stationary phase at pH 6.5, 8, and 8.5 and at pH 7 and 7.5. A new theory was presented to explain the beta-CD cavity structure balance between an ordered and disordered state. Variations of column temperature and mobile-phase pH tend to cause this phase transition between these two states, explaining the thermodynamic constant variations with pH and temperature.

The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X-FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin-dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase-promoting factor (MPF)-dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF-dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level.

Objective: To determine whether disruption of the cleavage site between protease and reverse transcriptase (RT) or the HIV-1 frameshift site could yield trans-dominant negative HIV-1 variants that interfere with wild-type viral replication., Design: Residues at the cleavage site between the HIV-1 protease and RT coding regions were mutagenized to produce a protease-RT (PR-RT) fusion protein that was expressed in the context of a full-length provirus. The PR-RT cleavage site mutation was also combined with a read-through mutation at the frameshift site in order to overexpress the mutant Gag-Pol polyproteins., Methods: COS-7 cells were transiently transfected with the mutant constructs to produce viruses harbouring the PR-RT fusion protein. In addition, we performed cotransfection studies in various cell types to analyze the inhibition of wild-type replication by the mutant constructs., Results: Immunoblot analysis revealed that this novel mutation prevented cleavage between the two proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. While both the protease and RT components of this fusion protein remained functionally active, viruses containing the cleavage site mutation were less infectious in tissue culture than wild-type viruses produced by COS-7 cells. This defect was further pronounced when the cleavage site mutation between protease and RT was overexpressed as a consequence of an additional mutation that interfered with frameshifting. Cotransfection of COS-7 cells with the mutant constructs and wild-type HIV-1 interfered with the replication of the latter and reduced the infectiousness of the virus particles produced., Conclusions: HIV-1 constructs harbouring a cleavage site mutation between protease and RT can probably act as trans-dominant negative mutants to interfere with wild-type viral replication.

Nonadrenergic imidazoline I2-binding sites colocalize with monoamine oxidase (MAO) in various tissues. As white adipocytes from various species have been reported to be very rich in I2-sites, the authors consider whether these cells show a substantial MAO activity and explore its functional role. Oxidation of [14C]tyramine by rat adipocyte membranes was dependent on both MAO and semicarbazide-sensitive amine oxidase (SSAO). Tyramine oxidation was identical in membranes and in intact adipocytes (Vmax: 11-12 nmol/min/mg protein). A similar effect of MAO and SSAO inhibitors was obtained in both the intact cells and the membranes: half of the activity was sensitive to semicarbazide and the other half more easily inhibited by MAO-A than by MAO-B inhibitors. As the reaction catalyzed by amine oxidases generates H2O2, which mimicks certain insulin effects in adipocytes, we tested whether tyramine oxidation influences glucose transport in adipocytes. One mM tyramine weakly stimulated glucose transport. A clear potentiation of tyramine effect occurred in the presence of 0.1 mM vanadate, ineffective by itself, reaching half-maximal insulin stimulation. This stimulation was sensitive to MAO and SSAO inhibitors and to catalase. The 5-fold activation of glucose transport was accompanied by translocation of GLUT4 transporters to the plasma membrane. This shows that tyramine is readily oxidized by adipocytes and potentiates the effects of vanadium on glucose transport through release of hydrogen peroxide. The role of the amine oxidases, which are highly expressed in adipocytes, allows them to be considered as more than mere scavengers of circulating amines.

Objectives: We wished to generate a number of genetic constructs containing mutations in the protease (PR) and reverse transcriptase (RT) genes of the Gag-Pol of human immunodeficiency virus type 1 (HIV-1) and to transfect these constructs into COS-7 cells to determine their effect on wild-type (wt) viral replication., Results: The mutated Gag-Pol polyproteins were incorporated into viral particles. Gag-Pol proteins that were mutated in PR as well as combinations of mutations in PR and RT inhibited the production of fully processed and infectious viral particles when these constructs were coexpressed with the infectious HIV-1 molecular clone pBH10. Viral particles produced after cotransfection of COS-7 cells with both pBH10 and infectious constructs containing Gag-Pol, mutated in PR alone or in both RT and PR, showed abnormal processing and lower infectivity. Complementation experiments in which pBH10 mutated in PR was coexpressed with wt Gag-Pol showed that the latter could be incorporated into the viral particles that were generated. COS-7 cells stably transfected with Gag-Pol, mutated in PR or in both PR and RT, and subsequently transfected with pBH10, produced levels of p24 and RT activity that were substantially diminished in comparison with levels produced by cells transfected with wt pBH10 alone., Conclusions: These results suggest that trans-dominant effects were potentially responsible for the observed inhibition of viral replication.

We have identified new members (X-PAKs) of the Ste20/PAK family of protein kinases in Xenopus, and investigated their role in the process that maintains oocytes arrested in the cell cycle. Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site, also deleted of its Nter-half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins, greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs induced apoptosis, as shown by activation of caspases and cytological changes in in vitro-assembled nuclei. This was suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart. Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus death by apoptosis and release of healthy oocytes from cell cycle arrest may be linked. That cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs.

Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

LEPTINE: Produced by adipose tissue, leptine is a regulatory hormone controlling body fat mass., Genetics: The human gene coding for leptine was first cloned in 1984. Its receptor, a member of the class 1 cytokine receptor family, has also been identified. VARIABLE SERUM LEVELS: Plasma levels of leptine in normal-weight subjects are in the 5ng/ml range and reach 50 ng/ml in obese subjects. Weight gain leads to higher blood levels and weight loss to lower levels. The effect of leptine is to approach a weight equilibrium. Leptine level is correlated with energy balance., Mode of Action: Leptine acts on hypothalamic centers controlling satiety. One of the essential mediators is neuropeptide Y. Resistance to leptine has been evidenced in human obesity. Resistance can occur as several levels and would be one of the explanations for massive obesity involving genetic factors. Leptine may also play a role in certain types of infertility.

Objective: To compare two contact and non-contact techniques using the VLAP system (visual laser ablation of the prostate)., Patients and Method: From January 1994 to April 1995, two groups of 50 patients were included in a randomized prospective study and were treated by the contact and non-contact laser technique, respectively. These patients presented with symptomatic BPH requiring transurethral electroresection. These patients were evaluated according to subjective (AUA score) and objective criteria (maximum flow rate, transrectal and suprapubic ultrasonography). ND:YAG laser energy was delivered by a Medilas 4100 source via a lateral beam fibre. Objective success criteria were an improvement of the AUA score by at least 50% and of the maximum flow rate by at least 50% with a difference of 4 mL/s. The failure criterion was the need to perform complementary electroresection during the first 6 postoperative months., Results: The mortality was 2% (cardiovascular causes), and the overall morbidity (first postoperative month) was 29%, with 21% of a vesical irritation syndrome, 6% of urinary tract infection and 2% of bladder clots. The global result at 12 months showed a statistically significant postoperative improvement of the subjective and objective parameters studied preoperatively (p = 0.0001). In the contact group, the AUA score, mean maximum flow rate and residual urine, which had preoperative values of 19.1, 9.1 mL/s and 141 mL, respectively, were equal to 2.3, 15.6 mL/s and 45 mL at 12 months. In the non-contact group, the same parameters, which had preoperative values of 17.8, 9.2 mL/s and 87 mL, respectively, were equal to 3.8, 13.3 mL/s and 47 mL at 12 months. The results, evaluated as a function of objective success criteria, showed a 12-month success rate of 68% for the contact method and 61% for the non-contact method. The mean hospital stay was 6 days and the mean duration of bladder catheterization was 5 days with the two techniques. During follow-up, 7% of patients required revision by electroresection, regardless of the technique used., Conclusion: this study demonstrated identical results for the contact and non-contact techniques using the VLAP system.

This study was designed to assess the efficiency of 2 kind of laser prostatectomy devices in the treatment of Benign Prostatic Hyperplasia: a non contact technique versus a contact technique versus a contact one. From January 1994 to September 1994, 100 patients were included in a randomized comparison of 2 laser prostatectomy devices with right angle firing laser fibers: a non contact technique with Urolase fiber (Bard) (50 patients) versus a contact technique with Fibertom fiber (Dornier) (50 patients). The Urolase fiber was used at 60 Watts power setting for 60 seconds and administered to each lobe at 2, 4, 8 and 10 o'clock positions. The Fibertom fiber was used by dragging or the so called "painting" technique at 3 and 6 months with 3 parameters: Madsen symptom scores, peak urinary flow rates and post-void residual urine volumes. Operative morbidity rate was 9%. No difference in morbidity between both fibers. No blood transfusion was required in any case. Statistical analysis of the aforementioned parameters shows a p-value of < 0.001 for all parameters. Comparing the 2 different fibers, there was no statistical difference in outcome for any of these parameters. From this study we conclude that the preliminary results achieved, using the Urolase and the Fibertom fiber, are equivocal and interesting. However, a long term follow-up is necessary to evaluate the definitive efficiency of laser prostatectomy and to determine the optimal procedure.

RNA2, the short segment of the genome of Dicenthrarchus labrax encephalitis virus (DIEV), a fish nodavirus causing seabass encephalitis, was cloned. Sequence analysis revealed that DIEV RNA2 contains a single open reading frame (ORF), which carries the catalytic D-75 residue but lacks the site for autocatalytic proteolysis, the process yielding the two capsid proteins of insect nodaviruses. Nevertheless, SDS-PAGE analysis of mature virions revealed a 43-45 kDa protein doublet. In order to determine the mechanism of synthesis of the two capsid proteins in DIEV, wild type and mutagenized forms of RNA2 were expressed in cell-free translation extracts and in transfected cells. Results showed that, despite the presence of the catalytic D-75 residue, the DIEV capsid protein doublet did not result from the assembly-dependent autocatalytic cleavage of a protein precursor. Moreover, our data show that, although suggested by sequence analysis, the DIEV capsid protein doublet results from neither an alternative initiation codon usage nor from a--1 ribosomal frameshift. Results of cell-free translation experiments demonstrate that the capsid protein doublet neither results of the proteolytic cleavage of a precursor nor of a degradation process. Kinetics of capsid protein synthesis in cell-free translation programmed with RNA2 revealed, instead, that the two capsid proteins are cosynthesized. Together these data strongly suggest that the DIEV capsid protein doublet results from cotranslational modification(s) of the ORF-encoded protein.

Specific interactions between the cell surface CD4 receptor and the HIV-1 envelope glycoprotein gp120 are responsible for the entry of HIV into host cells. Following infection, a down-modulation of CD4 at the cell surface is commonly observed. This may render cells resistant to subsequent infection by HIV as well as other viruses that also use CD4 as a portal of entry. This phenomenon is termed retroviral interference. CD4 down-modulation is complex and involves at least 3 viral gene products which include the envelope precursor gp160 and 2 auxilliary proteins Nef and Vpu. CD4 down-modulation has been observed in each of primary CD4+ T-lymphocytes and monocyte-derived macrophages, as well as both T and monocytic cell lines. CD4 down-regulation may occur at different levels. Specific binding of soluble gp120 may lead to internalization of CD4. The HIV-1 nef gene product which is expressed prior to HIV-1 structural proteins also causes the internalization of CD4 followed by its lysosomal degradation. During the late phase of viral gene expression i.e. viral structural protein synthesis, CD4-gp160 complexes forming in the ER represent another important factor leading to CD4 down-modulation. Finally, CD4 which is retained by gp160 in the ER, is specifically degraded in the presence of Vpu. Thus, it appears that CD4 down-regulation is of central importance to the life cycle of HIV-1.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the leukocyte-function associated antigen-1 and for Mac-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, phorbol myristate acetate, and retinoic acid. In this study, we investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in Cos-1 cells. Deletion mutant analysis provided evidence that a region located between -393 and -176 from the translational start site is critical to retinoic acid stimulation of luciferase activity. This region harbors the consensus sequence for a retinoic acid-responsive element (RARE) 5'-GGGTCATCGCCCTGCCA-3'. The Smal(-270)/Smal (-178) fragment containing this element conferred appropriate retinoic acid responsiveness to an enhancerless SV40 promoter. Cotransfection of expression vectors encoding the retinoic acid receptor alpha, beta, or gamma and retinoid X receptor alpha with reporter plasmids harboring the putative RARE demonstrated that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor beta/retinoid X receptor alpha-dependent fashion. Electrophoretic mobility shift assays showed that ICAM-1 and ADH3 RARE, a well-characterized RARE, display the same band shift pattern, bind retinoic acid receptor beta and retinoid X receptor alpha, and are mutually competitive.

The prolonged use of anti-viral nucleosides (ZDV, ddI, ddC) in HIV-infected patients has given rise to the isolation of viral variants that display resistance against these compounds. Tissue culture selection experiments, involving increasing concentrations of anti-viral compounds, have likewise been shown to select for drug-resistant strains of HIV. Cloning, sequencing and site-directed mutagenesis have shown that a series of point mutations in the viral reverse transcriptase (RT) are responsible for this phenomenon. A different series of mutations in RT are responsible for resistance against non-nucleoside inhibitors of this enzyme. These mutations are due to the error-prone nature of RT during viral replication. Mutated forms of recombinant RT, that derive from drug-resistant viruses, have reduced affinity for relevant triphosphorylated nucleosides.

Sequencing of the dnapol promoter region of Autographa californica nuclear polyhedrosis virus (AcNPV) revealed an overlapping open reading frame (ORF) in an antisense orientation, referred to as ORF-2. Analysis of the ORF-2 deduced amino-acid sequence revealed two short regions of homology with a similar ORF from Lymantria dispar nuclear polyhedrosis virus (LdNPV). Two 3' processing signals of this gene, expressed late during infection, were shown to be located on the orf-2 stop codon and 162 nucleotides further downstream.

Chromosome segregation is one of the most important acts in the life of the cell. Unequal inheritance of chromosomes (aneuploidy) is a cause of a number of disorders, particularly in humans, even though eukaryotic cells can arrest or delay the transition from metaphase to anaphase if an event critical to the completion of metaphase is impaired. In this report, we review recent advances in our knowledge of how the complex process of chromosome segregation is coupled with cell cycle progression, and starts at onset of anaphase with sister chromatids separation of the replicated chromosomes.

The present study was undertaken to examine a possible effect of aprotinin, a 6.5-kDa polypeptide with an inhibitory effect on proteolysis, on aminoglycoside nephrotoxicity. Experimental animals (female Sprague-Dawley rats, 175-200 g body wt) were treated for 4 days with 40 mg/kg gentamicin given ip at 12-hr intervals. Aprotinin (40,000 kIU per animal) was infused i.v. over a period of 8 days, using subcutaneously implanted miniosmotic pumps. In protocol A, infusion pumps were placed 4 days before starting gentamicin treatment. In protocol B, pumps were implanted 15-18 hr prior to first gentamicin administration. In addition to rats exposed to both gentamicin and aprotinin (GAP), animals were treated with gentamicin ip+saline i.v. (G), saline ip+aprotinin i.v. (AP), or received only saline by both routes of administration (C). All rats were terminated 4 days after the end of gentamicin dosing. One hour before sacrifice, 200 microCi of [3H]thymidine was given ip to each animal in order to monitor cell turnover in renal tissue. The kidneys were analyzed with respect to (i) histopathological alterations and renal dysfunction, (ii) aminoglycoside tissue accumulation, and (iii) tubular regeneration (measurement of cell proliferation). In animals receiving aprotinin alone, histological examination of renal cortex on paraffin sections disclosed mild tubular injury with focal cell necrosis. In plastic-embedded tissue, proximal tubule epithelium was characterized by the presence of numerous inclusions densely stained with toluidine blue. At the ultrastructural level, these inclusions appeared filled with amorphous electron-dense material. In gentamicin-treated animals, cortical drug accumulation reached values higher than 0.3 mg/g renal tissue, but a significant 30-40% decrease of gentamicin accumulation was noted in GAP groups, compared to G groups. Histological examination of renal cortex (paraffin sections) revealed the development of acute tubular necrosis in both G and GAP groups. Tubular injury was accompanied by mild renal dysfunction, as shown by the level of serum creatinine which was increased almost 3-fold in the G group, compared to C and AP groups. Aprotinin infusion produced a further increase of serum creatinine, particularly in protocol A where it was 72% higher for the GAP group than for the G group. In both G and GAP groups, postnecrotic tubular regeneration was evidenced by determining the rate of DNA synthesis and the frequency of S-phase cells in renal cortex. Both methods gave consistent results and showed a 8- to 13-fold increase of cell proliferation in groups receiving gentamicin alone, compared to C groups.(ABSTRACT TRUNCATED AT 400 WORDS)

The structurally unrelated immunosuppressants FK506 and cyclosporin A (CsA) act similarly, inhibiting a Ca(2+)-dependent signal required for interleukin-2 transcription and T-cell activation. Each drug binds to its cytosolic receptor, FKBP-12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In yeast, calcineurin has been implicated in recovery from alpha-mating factor arrest. Here we show that FK506 bound to yeast FKBP-12 appears to form a complex with yeast calcineurin. Moreover, recovery from mating factor arrest is highly sensitive to FK506 or CsA, and this sensitivity requires the presence of FKBP-12 or cyclophilin, respectively. These results define a key physiological target of an FK506- and CsA-sensitive signal pathway in yeast, suggest a high degree of mechanistic conservation with mammalian cells, and indicate that further examination of the yeast system should provide insight into the same process in T cells.

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23 degrees C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO(3) was added to the growth media. There were strong interactions between CaCO(3) and media, CaCO(3) and pH level, and CaCO(3), media, and pH level. In media with CaCO(3), all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO(3) increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 x 10/g). In CaCO(3)-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.

The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E. coli and is readily transferred between the two species by transfection and transformation. The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage. Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable. Some of the deletion mutants fail to lysogenize S. cattleya (Lyg- phenotype). The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis. The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S. cattleya.

The two aqueous solutions used for production of residential home insulation by the so-called urea-formaldehyde process were tested for their ability to react with cellular macromolecules. One of the components (the catalyst-surfactant) changed the apparent molecular weight of isolated DNA and increased its rate of attachment to bacterial and animal cells. The other component (the formaldehyde-urea resin) showed both these activities, especially following its exposure to mouse or rat liver extracts (postmitochondrial supernatants). Actively growing HeLa cells exposed to the catalyst-surfactant solution and then extracted with phenol yielded diminishing amounts of DNA, suggesting the formation of strong bonds to other cellular macromolecules, most likely to proteins. Formation of complexes between nucleic acids and proteins, enhanced cellular binding of DNA, and decreased extractability of DNA from growing cells exposed to chemicals have been found in separate studies to correlate with carcinogenic activity of various substances. Since a significant number of buildings will be insulated with this urea-formaldehyde foam and since such foam is also used in agriculture on crops, appropriate precautions should be taken to limit human exposure to the component materials.

The initial steps of adenovirus capsid morphogenesis and the sequence of entry of structural and nonstructural proteins into assembly-intermediate (IM) particles were investigated by pulse-chase labeling, temperature shifts, and cycloheximide inhibition of particle formation. The experiments were performed on wild-type and two assembly-defective, temperature-sensitive mutants, H2 ts 112 and H2 ts 107. The sequence of events in the adenovirus assembly can be schematized as follows. (i) Hexons, pentons, and protein IX assembled with scaffolding proteins 100K, PVIII, and PVII, precursor to the major core protein, to form a previral particle banding at a density of 1.285 in CsCl; (ii) additional incorporation of maturation and/or stabilization proteins IIIa, 50K, 39K, 28K, and PVI led to 1.295 IM; (iii) exit of 100K, 39K, and 28K, and entry of viral DNA gave rise to 1.370 IM; (iv) dephosphorylation and/or exit of 50K and exchange with core protein V and processing of precursors to VII, VI, VIII, and DNA-terminal protein resulted in formation of infectious 1.345 virion. The polypeptide composition of the new class of assembly-intermediate particles elicited by H2 ts 107 (1.285 IM), suggested that 100K, PVIII, and also PVII might serve as scaffold components for adenovirus capsid building.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Analysis of 100K-defective temperature-sensitive adenovirus mutants confirmed the multifunctional character of the nonstructural, virus-coded 100K protein. In addition to its function in hexon trimerization (altered in H5ts1), and its possible direct or indirect role in hexon transport to nucleus (mutated in H2ts118), genetic and biochemical evidence was presented that 100K play some critical role in the scaffolding process of adenovirus capsid. This function appeared to be defective in H2ts107 and to map between coordinates 69.0 and 69.9, leftward from the H5ts1 lesion (70-73 map units; Arrand, 1978). This corresponded to the central domain of the 100K protein, between amino acid 300 and 400 from the N end. DNA sequencing of cloned fragments of H2ts107 DNA overlapping the mutation revealed two point mutations on the same codon at nucleotide 25,082 and 25,083 (GAC----GCA), corresponding to a nonconservative amino acid change (aspartic acid----alanine) at position 324 in the 100K sequence. 100K of adenovirus 2 wild type (WT) was found to bind in significant amounts to novobiocin-affinity column, and to be coeluted with hexon, penton, IIIa, and cellular topoisomerase II activity, by novobiocin- or ATP-Mg2+-containing buffers. H2ts107 100K also bound to novobiocin column, but the elution pattern differed from that of WT, suggesting some alteration in the affinity of the mutated 100K for novobiocin. The same behavior on affinity column as H2ts107 100K was observed for 90K, a cleavage product of the 100K, found in great abundance in H2ts107 at 39.5 degrees and corresponding to the C-terminal moiety of the 100K molecule. This implied that the "novobiocin-binding" domain of the 100K was not confined at its N terminus, and was altered in the H2ts107 mutant.

Significantly enhanced attachment to Ehrlich ascites and Escherichia coli cells was observed for radioactive DNA and RNA in the presence of chemical mutagens and ultimate carcinogens. In some instances, formation of nucleic acid-protein adducts by these compounds further (or similarly) enhanced the binding. DNA irradiated with ultraviolet light in the presence of a protein bound more efficiently than either an unirradiated mixture of these two macromolecules or DNA irradiated alone. The spectrum of compounds tested and found active in this system includes alkylating agents, aromatic amines, and carcinogenic metals. Precarcinogens and nonultimate carcinogenic chemicals, as well as tumor-promoting agents, did not increase the binding. However, addition of extracts from mouse or rat livers activated precarcinogenic and proximate carcinogenic chemicals and resulted in enhanced cellular attachment of indicator nucleic acids in their presence. Possible usefulness of this test system for fast and efficient screening for environmental carcinogens and mutagens, as well as possible relevance of the observed phenomena to in vivo effects of chemical and physical carcinogens, is considered.