Enhancing fluorescence in vivo imaging using inorganic nanoprobes.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities.

[Revision of the European pharmaceutical legislation]. / Révision de la législation pharmaceutique européenne.

In accordance with the Regulation N 2309/93/EC, the Commission shall produce a synopsis of European registration operating procedures no later than 2001. On July 18, 2001, after a wide open consultation, the College of Commissioners adopted a Proposed Revision elaborated by the "DG Enterprises". This Proposed Revision was guided by the desire to facilitate access of citizens of the European Union to innovating and safe medications, to limit fragmentation of the interior market, prejudicial to the community pharmaceutical industry, and to prepare widening of the community. This synopsis focused essentially on registration procedures and pharmacovigilance, on patient information, on reform of the European agency for administrative protection of recorded data. The Proposed Revision is following the codecision procedure and should come into force, in a more or less amended form, at the end of 2004 at the latest.

A high frequency of sequence alterations is due to formalin fixation of archival specimens.

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Correlation of response to treatment and HIV genotypic changes during phase III trials with saquinavir and reverse transcriptase inhibitor combination therapy.

OBJECTIVES: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment. DESIGN: Virologic substudies of Phase III clinical trials NV14256 and SV14604. METHODS: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates. RESULTS: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV. CONCLUSIONS: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.

A colorimetric minisequencing assay for the mutation in codon 506 of the coagulation factor V gene.

We describe a colorimetric screening method for the mutation in codon 506 of the coagulation factor V gene. The nucleotide at the site of the FV:Q506 mutation is identified in an immobilized amplified DNA template by extension of a primer with a hapten-labelled dNTP using a DNA polymerase. The incorporated hapten is detected by an antibody-alkaline phosphatase conjugate that catalyses the formation of a coloured end product. The assay is carried out in a microtiter plate format, and the procedure is identical to that of enzyme immunoassays. It unequivocally identifies the FV:Q506 mutation in heterozygous and homozygous form. The colorimetric minisequencing method gave the same result as a 3H-based minisequencing assay and restriction site analysis with Mn11 used as reference methods. Because of its simple format and numeric result, the novel colorimetric minisequencing method should be an attractive alternative for screening for the FV:Q506 mutation in clinical laboratories.

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation.

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisiae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.

Effects of amino-acids on pancreatic hormones and gut glucagon-like immunoreactivity in the goose.

In the goose, alanine and arginine, intravenously or orally administered, act in the same way on pancreatic hormones; they both stimulate insulin and glucagon secretions. Conversely, whereas alanine treatment has no effect on plasma gut GLI, oral arginine stimulates gut GLI secretion. Since stimulation of gut GLI secretion does not occur with i.v. arginine, it may be assumed that this secretion depends on the intestinal transit of arginine and, as already described (Sitbon and Mialhe 1979), of glucose. The results, compared with studies on a similar species (duck) and on mammals, point out that i.v. infusion of alanine stimulates IRI and GLI secretions in the goose and not in the duck. In the same way, arginine i.v. infusion, contrarily to the observation made in the duck, is without effect on gut GLI secretion in the goose. Furthermore, insulin seems to be able to inhibit the alpha cell response to arginine infusion, as in mammals, whereas this is not the case in ducks.

Effects of total pancreatectomy and amino-acid treatment on plasma amino-acids and glucose in the goose.

1. Total pancreatectomy in the goose, in addition to the effects previously described (fatal hypoglycaemia and impaired tolerance), increases markedly the plasma amino-acid level. This increase corresponds to a significant rise in individually tested plasma amino-acids. In addition, pancreatectomy suppresses the hyperglycaemia which normally follows arginine treatment. 2. Oral administration of arginine induces hyperglycaemia in normal, but not in depancreatized geese. Therefore, the arginine effect upon the blood glucose level could be mediated by glucagon. Plasma glucose and glucagon changes, in normal animals with oral administration or i.v. infusion (Khemiss and Sitbon, 1982), agree with this conclusion. 3. Conversely, alanine elicits hyperglycaemia in normal animals, and such hyperglycaemia persists in pancreatectomized geese. Therefore, in the goose, in contrast to the duck, alanine appears to be a strong gluconeogenic amino-acid. These experiments with alanine indicate that closely related species, such as ducks and geese, may present important physiological differences.

[The endocrine pancreas of birds]. / Le pancréas endocrine des oiseaux.

The endocrine pancreas of birds contains 3 islet types and releases glucagon, insulin, somatostatin and avian pancreatic polypeptide (APP). Interactions between these hormones, and pancreatic hormone-plasma metabolite feedback mechanisms, are the main regulators of these secretions. In birds, total pancreatectomy induces a fatal hypoglycaemia associated with the disappearance of circulating glucagon, and an impaired glucose tolerance ascribed to the lack of insulin. Normoglycaemia can be restored in depancreatized birds by infusion of glucagon and insulin, provided that the G/I ratio is close to normal. Subtotal pancreatectomy provokes a diabetes characterized by a basal hyperglycaemia and an impaired glucose tolerance. However, while a transient diabetes is observed in most species, in the goose a permanent diabetes is obtained. These results point out the importance of the pancreas and especially of glucagon, in birds. This importance can also be detected in the protein metabolism, since total or subtotal pancreatectomy markedly increases the concentration of plasma aminoacids. As for lipid metabolism, in vivo and/or in vitro experiments have shown the sensitivity of the adipose tissue and liver cells to glucagon. Finally, contrary to the general opinion, insulin seems to be antilipolytic, as in mammals.

[Neonatal laryngeal paralysis. Course and treatment. 56 cases (author's transl)]. / Paralysies laryngées néo-natales: évolution et traitement. A propos de 56 observations.

56 laryngeal paralyses were seen in newborn infants between 1970 and 1976 (25 bilateral, 31 unilateral). The aetiology was obstetric trauma in 11 cases, nuclear agenesis in 7 cases, a severe neurological disorder (spina bifida, hydrocephaly, microcephaly, lesions of the central nervous system) in 13 cases, and congenital heart disease in 3 cases. In 10 cases, the paralysis was present in isolation. The initial state was not recorded in 11 cases. The course varied according to the aetiology: 5 deaths, 4 due to the severity of neurological problems. Regression, which invariably occured before the end of the 6th month, was seen in all the cases with an obstetric aetiology and in 50% of those in which the paralysis was present in isolation. There was persistence in the majority of neurological, nuclear or central causes. However, subsequent tolerance of persistent forms was good in all those patients followed-up on a regular basis, apart from 5 who underwent surgery. Treatment consisted of intubation for forms poorly tolerated initially, for the first few weeks. Tracheotomy did not prove necessary in any case. 5 patients underwent surgery: arytenoidectomy or arytenoidopexy via an extralaryngeal approach.

Pancreatic hormones and plasma glucose: regulation mechanisms in the goose under physiological conditions. II. Glucose-glucagon and glucose-insulin feed-back mechanisms.

We have studied the pancreatic hormone-glucose feed-back mechanisms by infusing glucagon (G), insulin (I) and glucose into normal fasting geese. The controls received saline. Whilst a NaCl 9% infusion is devoid of effect, the pancreatic hormones, used at physiological doses, modify the plasma glucose level, glucagon being hyperglycaemic and insulin hypoglycaemic. In addition, a physiological increase in plasma glucose provokes a drop in plasma glucagon and a rise in plasma insulin, thus a marked decrease in the G/I ratio. The results show that the pancreatic hormone-glucose feed-back mechanisms are effective under physiological conditions.

Pancreatic hormones and plasma glucose: regulation mechanisms in the goose under physiological conditions. I. Pancreatectomy and replacement therapy.

Measurements of plasma GLI and IRI in normal fasting geese, before and during constant I.V. infusion of saline, gave GLI/I ratios of 1.32 +/- .07 and 1.34 +/- .03 (w/w). As total pancreatectomy markedly reduces the pancreatic hormone level, leading to a mortal hypoglycaemia, we attempted to maintain plasma glucose within the normal range by constant I.V. infusion of glucagon and insulin into operated animals. The results as follows: 1. Blood glucose levels can be maintained within the normal range during experiments lasting 6 or more hours with a constant G/I ratio. 2. The G/I ratio obtained in operated animals (.96 +/- .12) is near to, but significantly lower (p less than .005) than, the GLI/I ratio measured in normal animals. This difference may be explained by the presence of a small amount of circulating gut GLI in the 2nd group.