Influence of gestational age on the EDIN score: an observational study.

BACKGROUND AND AIM: Hospitalised neonates, particularly if preterm, may be exposed to prolonged pain. At present the only validated scale to assess prolonged pain in preterms is the EDIN (Echelle Douleur Inconfort Nouveau-Né) scale. Gestational age has been shown to influence the response of infants to acute pain but its potential effect in the setting of prolonged pain has not been investigated. The aim of the present study was to evaluate whether neonatal maturity as expressed by gestational age and/or postnatal age influences their expression of prolonged pain. METHODS: In a 1 year period, 84 neonates (gestational age 25-41 weeks), referred to the authors' neonatal intensive care unit were evaluated using the EDIN scale two to three times a day (1571 scores). The EDIN scores were categorised as indicative (>6) or not indicative (< or =6) of pain. Gestational age and postnatal age were included in a logistic regression analysis along with some painful situations and analgesic treatment to identify the impact on the EDIN scores. RESULTS: Logistic regression analysis showed that the EDIN scores were positively associated with gestational age (odds ratio 1.166; 95% CI 1.123 to 1.211). Postnatal age, sepsis and presence of respiratory support also influenced the EDIN score. CONCLUSIONS: Gestational age influences expression of prolonged pain. Content validity of the EDIN scale could be improved by adding categories for gestational age and attributing higher basal scores to less mature newborns.

Quality control of 99Mo/99Tcm generators: results of a survey of the Radiopharmacy Working Group of the Italian Association of Nuclear Medicine (AIMN).

A multicentre survey of the quality control of 99Tcm generators has been completed: 245 generators from seven different commercial sources were tested over a period of 2 years. The results indicate that the mean pH of the eluates was 5.8 +/- 0.6; the aluminium contents were typically < 10 ppm; the radiochemical purity was 99.8 +/- 0.4% and the median 99Mo content was 3.8 x 10(-4) percent. The elution profiles gave a volume of 1.9 ml to obtain 50% of the total eluted activity and of 4.9 ml to obtain 95%. Other radionuclide impurities and heavy metal breakthrough were evaluated by graphite furnace absorption spectrometry and inductively coupled plasma mass spectrometry. National guidelines for the standardization of radiopharmacy procedures are currently being compiled.

Characterization of endoglucanase A from Clostridium cellulolyticum.

A construction was carried out to obtain a high level of expression in Escherichia coli of the gene celCCA, coding for the endoglucanase A from Clostridium cellulolyticum (EGCCA). The enzyme was purified in two forms with different molecular weights, 51,000 and 44,000. The smaller protein was probably the result of proteolysis, although great care was taken to prevent this process from occurring. Evidence was found for the loss of the conserved reiterated domains which are characteristic of C. thermocellum and C. cellulolyticum cellulases. The two forms were extensively studied, and it was demonstrated that although they had the same pH and temperature optima, they differed in their catalytic properties. The truncated protein gave the more efficient catalytic parameters on carboxymethyl cellulose and showed improved endoglucanase characteristics, whereas the intact enzyme showed truer cellulase characteristics. The possible role of clostridial reiterated domains in the hydrolytic activity toward crystalline cellulose is discussed.

Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA.

The nucleotide sequence of a Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCA)-encoding gene (celCCA) and its flanking regions, was determined. An open reading frame (ORF) of 1425 bp was found, encoding a protein of 475 amino acids (aa). This ORF began with an ATG start codon and ended with a TAA ochre stop codon. The N-terminal region of the EGCCA protein resembled a typical signal sequence of a Gram-positive bacterial extracellular protein. A putative signal peptidase cleavage site was determined. EGCCA, without a signal peptide, was found to be composed of more than 35% hydrophobic aa and to have an Mr of 50715. Comparison of the encoded sequence with other known cellulase sequences showed the existence of various kinds of aa sequence homologies. First, a strong homology was found between the C-terminal region of EGCCA, containing a reiterated stretch of 24 aa, and the conserved reiterated region previously found to exist in four Clostridium thermocellum endoglucanases and one xylanase from the same organism. This region was suspected of playing a role in organizing the cellulosome complex. Second, an extensive homology was found between EGCCA and the N-terminal region of the large endoglucanase, EGE, from C. thermocellum, which suggests that they may have a common ancestral gene. Third, a region, which extended for 21 aa residues beginning at aa + 127, was found to be homologous with regions of cellulases belonging to Bacilli, Clostridia and Erwinia chrysanthemi.

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli.

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.

Purification and partial characterization of two extracellular endoglucanases from Cellulomonas fermentans.

Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities.