Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

: The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.

A new gastric-emptying mouse model based on in vivo non-invasive bioluminescence imaging.

BACKGROUND: Different techniques were used to assess gastric emptying (GE) in small animals; most of them require sophisticated equipment, animal sacrifice and are expensive. In the present investigation a simple, non-invasive method based on bioluminescence imaging (BLI) is reported to study GE, using light-emitting Escherichia coli cells as a marker of the gastric content. METHODS: A new thermostable red-emitting luciferase was chosen as reporter gene to transform E. coli cells. Bioluminescent (BL) bacteria were administered to fasting mice, after a solid meal, and in response to different doses of metoclopramide (MET) and hyoscine butylbromide (HY). Bioluminescence imaging allowed to evaluate the real time 2D spatial and temporal distribution of bacteria along the gastrointestinal tract in animals and to calculate GE rate in basal conditions and following pharmacological stimulation. KEY RESULTS: The administered BL bacteria were easily imaged and localized in the stomach and subsequently followed in the duodenum and upper intestine allowing to accurately calculate GE. Gastric emptying after the test meal was significantly slower (T(1/2) 16 ± 3 min) than that obtained in fasting conditions (T(1/2) 2 ± 1 min); administration of HY (1 mg kg(-1) b.w.) significantly (P < 0.05) increased T(1/2) that was delayed up to 25 ± 4 min; MET (1 mg kg(-1) b.w.) significantly (P < 0.05) accelerated T(1/2), that was achieved within 8 ± 2 min. CONCLUSION & INFERENCES: The reported model is simple, inexpensive, reliable, sensitive and accurate; it can detect both acceleration and slowdown of GE. The model is useful in the investigation of new drug-induced alterations of gastric motility allowing to reduce the number of experimental animals.

Is cholangiocarcinoma another complication of insulin resistance: a report of three cases.

BACKGROUND: Cholangiocarcinoma is the second most common primary liver cancer, and the number of cases of intrahepatic cholangiocarcinoma (ICC) have been steadily increasing worldwide. Although the reasons for this surge are unknown, insulin resistance (IR) could be a risk factor, similar to what has been reported for other cancers. CASE REPORT: We report on 3 cases of ICC arising in subjects sharing IR as an underlying risk factor. Case 1 was an obese and dyslipidemic patient with NAFLD. The second and the third patients were affected by type 2 diabetes. CONCLUSIONS: Evidence for a link between IR and onset of cholangiocarcinoma in our patients rests on three lines of evidence: epidemiological, biological, and exclusion of others risk factors. Studies are needed to confirm our hypothesis that IR is a risk factor for the development of ICC.

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.

[Mini-invasive surgery of the retroperitoneal space in children]. / Chirurgie mini-invasive de l'espace rétropéritonéal chez l'enfant.

UNLABELLED: Although laparoscopic surgery now represents today an essential surgical technique, its use remains limited in urology and especially in pediatric urology for many reasons, main because of the lack of indications. After a large experience acquired in abdominal laparoscopic surgery, and because we were convinced of the advantages offered by this new mini-invasive approach, we have tried to develop it for the retroperitoneal space. METHODS: Over a five-year period we performed 88 retroperitoneal procedures in children:--50 nephrectomies (44 total, 10 partial) for the following indications: 15 polycystic dysplastic kidney, 13 kidney destroyed by reflux, 18 by obstruction, hypertensive uropathy 3, pyonephrosis 1.--5 renal cystectomies, 3 pyelolithotomies, 2 pyeloureteral obstructions, 2 adrenalectomies, 1 retrocaval ureter, 25 varicoceles. The age range was 2 months to 16 years (mean: 3.7 years, 25 children under 1 year). The patients were placed in the lateral debubitus. The retroperitoneal space was created by dissection under direct vision, then insufflation was performed directly in the retroperitoneal space without balloon. Three or four ports were used except for varicocelectomy which was performed with only one port and an operating channel telescope. RESULTS: Follow-up range was 6 months to 5 years. The mean operating time was 96 minutes (35 to 210 min.). Average postoperative stay was 2 days. Conversion was needed in 7 cases (8%). Operative incidents consisted of one duodenal perforation, one ureteral burn, 21 peritoneal perforations (24%). There were 5 postoperative complications (2 urinomas after partial nephrectomy, 1 hydrocele, 1 varicocele recurrence, 1 recurrent stones) not related to the technique. 3 cases needed reoperation (ureteral injury, varicocele recurrence, recurrence of cystine stones) with good result. CONCLUSION: Like other laparoscopic techniques, retroperitoneoscopy requires a training: it remains delicate in children because of the reduced working space and the fragility of the peritoneum. However the advantages seem sufficiently obvious for us to recommend and promote this procedure.

The unique chaperone operon of Thermotoga maritima: cloning and initial characterization of a functional Hsp70 and small heat shock protein.

The hyperthermophilic eubacterium Thermotoga maritima possesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of T. maritima, one of the deepest-branching eubacteria known.