Structural enhancement and video endoscopy: results of a large prospective comparative study.

INTRODUCTION: The structural-enhancement (SE) function electronically improves the video-endoscopic signal of Olympus processors (EXERA CV-160 or greater), enabling an increase in relief that may help in the detection of flat or ulcerated and raised lesions, especially those of small size. We assessed the diagnostic impact of this technique in the screening of lesions during basic video colonoscopy. METHODS: Maximum-level SE was programmed into processors on alternate weeks, and endoscopy dates were planned by an assistant unaware of the SE schedule, thus ensuring randomization. The endoscopists-senior practitioners with 3-29 years of digestive endoscopy practice-were informed of the experiment >3 weeks before it began and were not told about it again either before or during the study. This was to ensure that endoscopy examinations were performed without over-awareness of the technical conditions. GIF-100 to -160 Olympus endoscopes were used. RESULTS: During the study, 606 patients underwent upper digestive video-endoscopy, 305 with and 301 without the use of the SE function. Of 645 patients who underwent video colonoscopy, 593 were included in the study and 52 were excluded due to poor cleansing (8%); of those included, 330 were analyzed with and 263 without the SE function. We observed no differences in the detection of lesions (small or large) by either upper digestive endoscopy or video colonoscopy. CONCLUSION: This is the first study comparing video-endoscopy diagnosis with or without SE during upper digestive endoscopy and colonoscopy. The SE function available on Olympus video-endoscopy processors had no impact on the detection of lesions, not even on those of very small size.

Antiviral activity and cross-resistance profile of P-1946, a novel human immunodeficiency virus type 1 protease inhibitor.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

Wild type human p53 is antiproliferative in SV40-transformed hamster cells.

The transformation related protein p53 has been implicated in the process of normal cell proliferation and neoplastic transformation. In this study, the influence of wild type human p53 on cell proliferation was examined. Plasmid constructs encoding the wild type human p53 and various mutant p53 cDNAs, driven by the mouse mammary tumor virus (MMTV) promoter linked to the dominant biochemical selection marker gpt, were used in a colony forming assay employing SV40 transformed HR8 hamster cells. Plasmids encoding wild type p53 drastically reduced the number of gpt+ colonies obtained after transfection, whereas the mutant forms of p53 had no effect. Stable clonal hamster cell lines that constitutively express wild type p53 were isolated and found to have altered growth characteristics (i.e. lower saturation densities, increased doubling times). These findings are consistent with the notion that wild type p53 protein could function as a growth suppressor. The potential role of p53 in the normal cell cycle and in the transformation process is discussed.

Antiviral properties of simple difunctionalized enols targeted to the HIV-1 protease.

The human immunodeficiency virus type 1 (HIV-1) protease catalyses the specific cleavage of the virion structural polyproteins p55gag and p160gag-pol and is, therefore, essential for viral maturation. We have previously reported a series of low molecular weight non-peptidic enol-based compounds that inhibit the HIV-1 protease activity in a competitive fashion (Vaillancourt et al., Bioorg. Med. Chem., 2 (1994) 343-355). Here we demonstrate that VS-215 and VS-261, two of these non-peptidic inhibitors, impair viral polyprotein maturation and exhibit antiviral activity in infected MT4 cells. The ID50 for these two compounds ranged between 24 and 50 microM whereas their TD50 ranged between 60 and 200 microM depending on the cell lines used. The calculated therapeutic index of these two inhibitors both had values of 2.5 even though they were shown to be non cytotoxic at their ID50. Their calculated permeability index ranged between 0.09 and 0.79 suggesting that these enol-based inhibitors efficiently reach the site of protease activity. These results provide new information on the therapeutic potential of this new class of protease inhibitors and emphasize the usefulness of enol chemistry in the development of anti-HIV-1 protease inhibitors.

Characterization of mutant HIV-1 integrase carrying amino acid changes in the catalytic domain.

To gain insight into the importance of conserved residues in the core domain of HIV-1 IN, we performed site-directed mutagenesis of the full-length enzyme, overexpressed the mutant proteins in E. coli, purified and analyzed their 3'-processing, integration and disintegration activities in vitro. Change of E152V in the DD(35)E motif abolished all detectable activities of IN. Alteration of two highly conserved residues, P145 and K156, by isoleucine, resulted in a substantial loss or completely abolished the three activities of the enzyme. Mutant P90D weakly reduced the 3'-processing but severely affected the two other IN activities. Results obtained from double and triple mutations, P90D/P1451 and P145I/F185K/C280S, clearly suggest a crucial role of P145 in the catalytic function of IN, whereas the mutants V150E, L158F and L172M had no detectable effect on any of the IN activities. Taken together, these results allowed us to conclude that all the conserved amino acids in the core domain of IN are not equally important for catalytic functions: like D64, D116 and E152, our data suggest that P90, P145 and K156 are also essential for all three enzymatic activities of HIV-1 IN in vitro, whereas V150, L158 and L172 appear to be less critical.

Analysis of the DNA substrate structure and number of the processing sites on the activities of HIV-1 integrase in vitro.

A series of DNA substrates were synthesized to analyze the 3'-processing, integration and disintegration reactions taking place concurrently on the same DNA molecules and to evaluate the potential effects of various structural modifications of these molecules on the activities of HIV-1 integrase (IN). Our results indicate that DNA substrates containing multiple recognition sites for IN can produce efficiently the three activities of the enzyme. The 3'-processing and disintegration sites are recognized and processed by IN, both reactions being carried out in a competitive manner by the enzyme on the same DNA molecule. The presence of the gaps and unpaired nucleotides in the region surrounding the disintegration site had major deleterious effects on enzymes disintegration activity. Analysis of a different conformation at the base of the DNA hairpin has revealed a significant improvement of IN disintegration activity in the presence of double-stranded DNA on the 3' side of the disintegration site, suggesting that this region plays an important role in the stability of the enzyme-substrate complex. Interestingly, the efficiency of disintegration was strongly diminished in the presence of an unpaired nucleotide located immediately at the 3' end of the cleavage site. Overall, our results underline the extreme sensitivity of the HIV-1 IN to its substrates structure and conformation, especially for its disintegration activity, and the considerable importance of the disintegration activity in the reactions carried out in vitro by the purified enzyme.

Solution structure of the HIV protease inhibitor acetyl-pepstatin as determined by NMR and molecular modeling.

The structure of acetyl-pepstatin has been investigated in solution by two-dimensional NMR spectroscopy and molecular modeling. The analysis of DQFCOSY, TOCSY and NOESY spectra lead to a full assignment of the -NMR signals both in DMSO-d6 and in TFE-d3:H2O 1:1. Interproton distances, dihedral angles and exchanger regimes of NH or OH protons were derived from ROESY connectivities, coupling constants and temperature dependences of the chemical shifts, respectively. Molecular modeling using the NMR distance and dihedral angle constraints obtained in DMSO-d6 yielded a model showing a well-defined structure for the N-terminal segment Ac-1 to Sta-4, but a flexible structure for the C-terminal segment. The structure was less defined in TFE-d3:H2O 1:1 and 13C T1 measurements are indicative of higher mobility. Comparison of the NMR-determined solution structure of acetyl-pepstatin with its crystal structure when bound to HIV-1 protease shows that the conformation is more extended in the complex as a result of intermolecular interactions.

The impact on clinical practice of endoscopic ultrasonography used for the diagnosis and staging of pancreatic adenocarcinoma.

CONTEXT: Endoscopic ultrasonography is considered a highly accurate procedure for diagnosing small pancreatic tumors and assessing their locoregional extension. OBJECTIVE: To evaluate the impact of endoscopic ultrasonography on the management of pancreatic adenocarcinoma in clinical practice. PATIENTS: Sixty-four consecutive patients (mean age 70.5 plus/minus 11.9 years) hospitalized for staging or diagnosis of pancreatic adenocarcinoma were retrospectively (from January 1995 to November 1997) or prospectively studied (from December 1997 to August 1999). SETTING: Group 1 consisted of 52 patients with pancreatic adenocarcinoma which was discovered using computerized tomography scanning and/or ultrasound. Endoscopic ultrasonography was utilized for staging purposes only in patients who were considered to be operable and the tumor to be resectable based on computerized tomography scanning criteria. Group 2 consisted of 12 patients who were diagnosed as having a pancreatic adenocarcinoma using endoscopic ultrasonography whereas computerized tomography scanning and ultrasound was negative. MAIN OUTCOME MEASURES: The impact of endoscopic ultrasonography was analyzed on the basis of the number of patients requiring endoscopic ultrasonography as a staging procedure (Group 1) and by evaluating the performance of endoscopic ultrasonography in determining resectability (Groups 1 and 2) based on the surgical and anatomopathological results. RESULTS: Endoscopic ultrasonography was performed in 20 out of 64 patients (31.3%): 8/52 in Group 1 (15.4%) and all 12 patients of Group 2. Endoscopic ultrasonography correctly assessed an absolute contraindication to resection in 11 cases. Resection was confirmed in 8 of the 9 cases selected by endoscopic ultrasonography. The positive predictive value, negative predictive value and overall accuracy of endoscopic ultrasonography for determining resection were 89%, 100%, and 95%, respectively. CONCLUSIONS: The impact of endoscopic ultrasonography seems especially relevant for the detection of pancreatic tumors after negative computerized tomography scanning, and for the prevention of unnecessary laparotomies as complementary staging after ultrasonography and computerized tomography scanning.

Lipid-based delivery of combinations of antisense oligodeoxynucleotides for the in vitro inhibition of HIV-1 replication.

We evaluated a new approach to AIDS therapy by using combinations of oligodeoxynucleotides (ODNs), delivered with a lipid-based carrier system, that target different HIV viral genome sites. We identified some of the factors that seem to influence the effectiveness of a combination strategy in cell cultures including ODN concentrations, type of infection (acute vs chronic), backbone modification of the ODN, and the number of sequences. When delivered by the DLS carrier system, some advantages of using a combination of ODNs over treatment with only one ODN could be observed in acute infection assays but not in the chronic infection model. These results suggest that in the acute infection model, the 3 different antisense ODNs in the "cocktail" might block an early step of virus replication by combined inhibitory effects. Various combinations of phosphorothioate-modified (PS) and unmodified oligonucleotides delivered by the DLS system were compared for their antiviral activity in a long-term acute assay using HIV-1 (IIIB strain)-infected MOLT-3 cells. The most effective combination had 3 phosphorothioate antisense ODNs: Srev, SDIS, and SPac (>99% inhibition at 100 pM). However, the additive effect determined when using ODN combinations was rather low, revealing the high level of nonsequence specificity in HIV-1 cell culture models. Data illustrated the high sequence nonspecific activity of ODNs, especially when comparing activity of antisense ODNs with activity of random control sequence ODNs. The latter exhibited an inhibitory effect similar to that of antisense ODNs under our experimental conditions. Nevertheless, we demonstrated that it is possible to achieve high anti-HIV activity by using, in combination, picomolar range concentrations of antisense oligonucleotides complexed to a lipid-based carrier system such as the DLS system, without increasing cell toxicity.

Sequence analysis of the human thymidine kinase gene promoter: comparison with the human PCNA promoter.

We report the sequence of 4264 nucleotides of 5' flanking sequence of the human thymidine kinase gene, a gene that is maximally expressed at the G1/S boundary of the cell cycle. The position of nucleotide sequences which can act as binding sites for trans-acting factors, Sp-1, AP-1/jun, AP-2, OTF-1 and CAAT box factors as well as other potential cis-acting sequences have been mapped. The organization of these cis-acting sequences in the promoter of the human PCNA gene (another gene that is maximally expressed at the G1/S boundary) are shown for comparison. The potential role that these sequences may play in the transcriptional regulation of these genes is discussed.

[Trends for the management of pancreatic adenocarcinoma in a hospitalized-based population (1980-1994)]. / Evolution de la prise en charge de l'adénocarcinome pancréatique dans une série hospitalière (1980-1994).

OBJECTIVE: To determine the trends for the management of pancreatic adenocarcinoma between 1980 and 1994 in a hospital-based population. METHODS: Data of patients from the Doubs department and hospitalized for pancreatic adenocarcinoma in the University Hospital of Besançon were analyzed. Two study periods were determined: 1980-1989 and 1990-1994. RESULTS: One hundred and thirty five cases were diagnosed (77 male, 58 female, mean age 65.6 +/- 12.1 years). Weight loss (62.3%), pain (52.7%) and jaundice (43.3%) were the most frequently reported symptoms. Their proportion and their duration were similar according to the period. Diagnosis of the pancreatic tumor was made by ultrasonography or tomodensitometry in 97.8% patients. Distribution of tumors according to the AJCC staging was unchanged during the study. Pre-operative screening frequently underestimated tumor stage, even in the more recent period. The rate of complete resection (18.5%) did not increase. The 5-year actuarial survival rate (3.05%) remained unchanged. A metastatic spread represented the only independent prognostic factor. CONCLUSIONS: The management and the prognosis of pancreatic adenocarcinoma have not fundamentally changed. Recent imaging techniques should improve staging accuracy. Advances in adjuvant therapies represent a major issue for the future.

[Repetitive acute pancreatitis in a late-diagnosed cystic fibrosis: prevention of relapses by octreotide in the long term]. / Poussées à répétition de pancréatite aiguë dans une mucoviscidose à révélation tardive: prévention par octréotide au long cours.

We report on the case of a 35 year-old woman who was initially admitted for acute pancreatitis in october 1995. The patient was suffering from asthma (since childhood) and diffuse abdominal pain (since adolescence). The diagnosis of cystic fibrosis was made fortuitously during a sterility evaluation. After extensive etiological screening the acute pancreatitis was considered to be a manifestation of the cystic fibrosis. Despite therapy with pancreatic enzymes, the patient continued to suffer from chronic abdominal pain. High intake of analgesics was required. Until December 1995, the patient was repeatedly admitted for episodes of acute pancreatitis. In January 1996, we initiated a preventive treatment with subcutaneous octreotide between 100 and 200 microgram, three time a day. Thereafter, there were fewer episodes of pancreatitis and the consumption of analgesics decreased. Side effects of octreotide were intermittent diarrhea and development of cholelithiasis that was complicated by biliary migration in November 1998. In June 1999, the prolonged-release form of the molecule was given without modification of the efficacy.

[Histamine-producing neuroendocrine tumor of the ampulla of Vater]. / Tumeur neuro-endocrine de l'ampoule de Vater sécrétant de l'histamine.

Neuroendocrine tumors of the ampulla of Vater are rare (less than 100 cases reported). We report here a new case characterized by histamine secretion, a hitherto unreported feature. Clinical presentation is similar to that of other tumors of the ampulla of Vater. In our observation, the patient had noticed urticaria on the right forearm for several months. Tumor of the ampulla was confirmed by endoscopic ultrasonography, while neuroendocrine characterization was assessed on biopsies after endoscopic sphincterotomy. Histamine concentration in blood was the only elevated neuroendocrine marker and returned to normal after surgical resection. Histology showed a neuroendocrine tumor with extension to lymph nodes. On immunohistochemical analysis, production of histamine was confirmed, and the diagnosis of mastocytoma was eliminated. In view of the literature, neuroendocrine tumors of the ampulla of Vater are associated with a good prognosis (5 year-survival rate: 90%) despite early lymph node involvement.

A primer on insulin use.

Insulin treatment, if thoughtfully designed and carefully monitored, can allow the diabetic patient to lead a close-to-normal life. Several types of insulin with varying times of action are available, and regimens can be tailored to a patient's needs and life-style, with alterations made for such things as time changes, exercise, and fasting for surgical procedures. Self-monitoring of blood glucose levels can provide patients with an important measure of independence, as can instruction on recognizing and correcting episodes of hypoglycemia. Insulin is an important part of managing ketoacidosis and hyperosmolar hyperglycemia syndrome, although success depends on overall care.

Characterization of dynamic state inhibitors of HIV-1 protease.

The inhibitory behavior of simple N-protected amino acids containing a dysfunctional enol group was analyzed. Pseudo-Hill coefficients below 0.5 were obtained for the enol compounds. The active moiety of the enols described herein is involved in an equilibrium between keto and enol forms. The enol in its E form interacts strongly with the enzyme and we suggest that the Z form is expelled from the active site, as suggested by molecular modeling studies using the docking technique. Also, tetrahedral intermediate mimicry is achieved by the enol form, but not by the corresponding keto form. A pseudo-Hill coefficient of 0.25 has been determined for the Boc-phenylalanine enol, and by multiplying the concentration of inhibitor by its pseudo-Hill coefficient, a Ki of 0.82 microM has been determined for the inhibitory E-enol form. Boc-tyrosine enol generated a Ki of 0.39 microM. The requirements at the P1' position to achieve good inhibition are also described.

Synthesis of novel inhibitors of the HIV-1 protease: difunctional enols of simple N-protected amino acids.

A series of enol HIV-1 protease inhibitors which show competitive inhibition and the structure-activity relationship study which led to the design of these compounds are reported. By systematically modifying simple amino acids, Boc-Phe enol and Boc-Tyr enol derivatives yield nanomolar Kiapp values (Kiapp = 0.485 microM and Kiapp = 0.425 microM, respectively). These enols are of low molecular weight (< 500 g/mol) and of non-peptidic nature. The enols are synthesized in a one step chemical synthesis and modifications to increase their potency could easily be performed. Boc-Phe enol and Boc-Tyr enol showed low inhibitory effect on pepsin, Kiapps of 23 and 149 microM, respectively, and Boc-Phe enol showed a Kiapp of 20 microM for cathepsin D. Neither of these two compounds inhibited renin (< 10% inhibition at 200 microM).

Some remarkable effects of thiopeptide and derived linkages on lysozyme release from neutrophils by esters of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe-OR).

A variety of recently synthesized analogues of the chemotactic agent f-Met-Leu-Phe-OR modified in the backbone were tested for their ability to induce the release of lysozyme from human neutrophils. In sharp contrast to the effects of thiopeptide linkages on the biological activity of Leu5-enkephalin as previously reported, the presence of single thioamide bonds at either one of the endo-positions of the chemotactic peptide, abolished activity. Thioamide-derived linkages such as amidoximes and cyanamidines were generally also detrimental to activity, except in the cases of the cyanamidoformyl derivatives which showed enhanced activity and two amidoxime esters, one O-acetylated and the other O-esterified intramolecularly, which retained moderate activity. The mechanistic significance of these results is discussed in terms of conformational effects on receptor recognition.

A competitive enzyme immunoassay for albuterol: its application for the drug screening in urine.

A competitive enzyme immunoassay using purified monoclonal IgG1 and an alkaline phosphatase-albuterol derivative has been developed for the quantification of albuterol in urine. The calibration curve obtained in optimal incubation conditions is characterized by a minimum detectable level of 26 fmol/well and a working range from 52 fmol to 4,2 pmol/well. This method allows the precise and accurate quantification of albuterol in horse urine without any clean up or extraction step. Moreover the definition of its specificity shows a cross-reactivity of the antibody with the glucurono-/sulfo-conjugates of albuterol. This property is particularly interesting for the screening of urinary albuterol residues in meat producing animals.

Isolation of a human sequence which complements a mammalian G1-specific temperature-sensitive mutant of the cell cycle.

We have isolated a human genomic sequence which corrects the temperature-sensitive phenotype of the G1-specific cell cycle mutant ts13. Transfection of a thymidine kinase negative (TK-) derivative of ts13 with high molecular weight human DNA from placenta of HeLa cells resulted in the isolation of thermocompetent transformants. These transformants were found to possess discrete Hind III fragments containing human Alu family sequences which were conserved in several independent secondary transformants. A transforming sequence was recovered from a HindIII recombinant bacteriophage library prepared from the DNA of one such secondary transformant. The transforming sequence was isolated as a 12.5 kb Hind III fragment.