[Hospital clinical records accuracy in traceability of healthcare associated infections]. / Accuratezza della documentazione clinica ospedaliera nella tracciabilità delle infezioni correlate alle pratiche assistenziali.

We conducted a retrospective analysis on health care records in order to validate its accuracy in the reporting of healthcare associated infections (HAIs) for the purpose of supporting epidemiological surveillance and nursing-sensitive patient outcomes studies. The health care records have been selected on the basis of the database of alert microorganisms in a teaching Hospital of North-Eastern of Italy, for the years 2005-2006-2007 in three wards (Hematology, ICU and Surgical ward). In 80/107 (74.8%) cases of alert microorganisms a written record was found in the patient's health care records, most frequently in the nursing records (64/80, 80%). In the health care records have been reported 21 diagnosis of infection (21/107, 19.6%). The presence of written symptoms was heterogeneous among the different sources considered (medical and nursing records, vital parameters and therapy sheets). The results are not completely satisfactory from the point of view of the information accuracy. The promotion of integrated clinical health care record systems (doctors/nurses), also electronics, a more accurate compilation and periodical supervision would be needed.

Vaccination against contagious bovine pleuropneumonia and the use of molecular tools in epidemiology.

Contagious bovine pleuropneumonia is a serious threat to cattle not only in Africa but also in southern Europe and possibly Asia. It is now present in countries that had been free of the disease for many years, giving rise to doubts about the efficiency of the control strategies. In Africa CBPP is controlled mainly by a vaccination policy that uses variant strains of Mycoplasma mycoides subsp mycoides biotype SC, called T1/44 or T1sr. Until recently, it was not possible to differentiate the various strains within the biotype and consequently to identify the vaccine strains. Restriction analysis of mycoplasma DNA with HindIII and Pst1 has been applied to 24 strains of African origin and one European strain. Each enzyme gave rise to different restriction profiles and the combination of the results permitted subdivision of these strains into 9 groups. Interestingly, some profiles of pathogenic strains seem to be restricted to certain geographical areas. The profile of the poorly immunogenic vaccinal strain KH3J is also very peculiar, and it is easily distinguished from that of the other vaccine strains originating from T1. This technique is simple once the strains are isolated. Efforts are now under way to use molecular tools based on PCR products to alleviate the difficulty of isolation.

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide.

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.

Phylogeny of the Mycoplasma mycoides cluster as shown by sequencing of a putative membrane protein gene.

The Mycoplasma mycoides cluster is made of six species that are closely related both genetically and phenotypically. Two are of particular importance, M. mycoides subsp. mycoides SC causing contagious bovine pleuropneumonia and M. capricolum subsp. capripneumoniae causing contagious caprine pleuropneumonia. The sequences of a putative membrane protein gene and partial flanking open reading frames have been obtained from various strains in this cluster, including all reference strains. Sequence analysis showed this locus is present and fully conserved in all strains of M. mycoides subsp. mycoides SC isolated from geographically most distant places worldwide. In M. capricolum subsp. capripneumoniae polymorphism in this locus has been found at seven positions and revealed that they can be used as epidemiological markers. Conserved regions were used to define a primer pair that enables the amplification by PCR of two fragments 302 and 1298bp long, respectively. The 302bp long fragment contains an intergenic sequence that can be used for phylogenetic studies or for identification purposes. Parsimony analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides cluster into two subgroups that is in accordance with results obtained by phenotypic methods. Two lineages exist within the capricolum subgroup, one of them clustering strains identified as M. capricolum subsp. capricolum, M. capricolum subsp. capricolum and M. sp Bovine Group 7. However M. capricolum subsp. capripneumoniae strains can readily be identified by three specific nucleotide positions or by sequencing the 1298bp long fragment. There is no clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides subsp. mycoides LC and M. mycoides subsp. capri should not be separated into two subspecies. Mycoplasma mycoides subsp. mycoides SC strains can easily be distinguished as they bear an insertion sequence 15bp downstream from the stop codon of the membrane protein gene.

A specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP).

Contagious caprine pleuropneumonia is a severe infectious disease of goats in Africa and the Middle East. It is caused by a fastidious mycoplasma, Mycoplasma capricolum subsp. capripneumoniae, a member of the "M. mycoides cluster". Members of this cluster share genomic and antigenic features, which result in common biochemical and serological properties, complicating species identification. Two species of this cluster, M. mycoides subsp. capri and M. mycoides subsp. mycoides large colony biotype, are very often isolated from clinical cases resembling contagious caprine pleuropneumonia. Furthermore, in the laboratory, M. capricolum subsp. capripneumoniae can be easily confused with the closely related capricolum subspecies. Considering these constraints and the scarcity of available methods for identification, a specific polymerase chain reaction was developed. A DNA fragment of 7109 bp containing genes coding for the arginine deiminase pathway (ADI) was chosen as target sequence for the selection of a specific primer pair. The full ADI operon from M. capricolum subsp. capripneumoniae strain GL100 was sequenced. Polymorphism within this locus was analyzed by comparison with the sequence from the closely related IPX strain (M. capricolum subsp. capricolum). It varied from 0.6% to 3.5%. The highest divergence was found in a region coding for arcD. Therefore, this gene was chosen as target for the specific amplification of a 316 bp-long DNA fragment. The specificity of this PCR was validated on 14 M. capricolum subsp. capripneumoniae strains and 27 heterologous strains belonging to the "M. mycoides cluster" and M. putrefaciens. This new PCR will be a valuable tool for the surveillance of contagious caprine pleuropneumonia.

Molecular epidemiology of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides subspecies mycoides biotype SC strains.

Contagious bovine pleuropneumonia is a bacterial disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), and included in list A of the Office International des Epizooties. It is one of the major constraints to cattle raising in sub-Saharan and south-western Africa and also a threat to all countries currently free of the disease. MmmSC strains were considered very homogeneous until 1995, when various techniques such as enzymatic restriction of whole DNA or Southern blotting showed that this was not the case. These techniques are unfortunately difficult to standardize and require the extraction of DNA from an MmmSC culture. We therefore decided to investigate the possibility of constructing a molecular epidemiology tool based on multilocus sequence analysis (MLSA) with PCR amplification of various loci followed by sequencing. Six loci were found suitable for this purpose and an additional PCR was designed to detect the presence of an 8.8kb deletion described by others in some strains. Fifteen different MLSA profiles were evidenced in our study. They allowed a clear distinction between European, south-western African and sub-Saharan strains. In addition, the results obtained on strain PO1967 confirmed its European origin, even though it does not exhibit the 8.8kb deletion. This new tool for contagious bovine pleuropneumonia may prove particularly useful for identifying MmmSC strains in countries at risk from contamination. It can also easily be refined by adding more strains or other loci of interest.

Genetic evolution of Mycoplasma capricolum subsp. capripneumoniae strains and molecular epidemiology of contagious caprine pleuropneumonia by sequencing of locus H2.

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in developing countries. Its exact distribution is not well known, despite the fact that new diagnostic tools such as PCR and competitive ELISA are now available. The authors developed a study of the molecular epidemiology of the disease, based on the amplification of a 2400 bp long fragment containing two duplicated gene coding for a putative membrane protein. The sequence of this fragment, obtained on 19 Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from various geographical locations, gave 11 polymorphic positions. The three mutations found on gene H2prim were silent and did not appear to induce any amino acid modifications in the putative translated protein. The second gene may be a pseudogene not translated in vivo, as it bore a deletion of the ATG codon found in the other members of the "Mycoplasma mycoides cluster" and as the six mutations evidenced in the Mccp strains would induce modifications in the translated amino acids. In addition, an Mccp strain isolated in the United Arab Emirates showed a deletion of the whole pseudogene, a further indication that this gene is not compulsory for mycoplasma growth. Four lineages were defined, based on the nucleotide sequence. These correlated relatively well with the geographical origin of the strains: North, Central or East Africa. The strain of Turkish origin had a sequence similar to that found in North African strains, while strains isolated in Oman had sequences similar to those of North or East African strains. The latter is possibly due to the regular import of goats of various origins. Similar molecular epidemiology tools have been developed by sequencing the two operons of the 16S rRNA gene or by AFLP. All these various techniques give complementary results. One (16S rRNA) offers the likelihood of a finer identification of strains circulating in a region, another (H2) of determining the geographical origin of the strains. These tools can make a very useful contribution to understanding the epidemiology of CCPP.

Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis.

Streptomyces pristinaespiralis synthesizes pristinamycin, a member of the streptogramin antibiotic family which consists of a mixture of two types of chemically unrelated compounds named pristinamycins I and pristinamycins II. In order to estimate the size of the Strep. pristinaespiralis chromosome and to elucidate the organization of the pristinamycin biosynthetic and resistance genes already identified, it was decided to use the pulsed-field gel electrophoresis technique. Results indicate that the Strep. pristinaespiralis chromosome is linear and about 7580 kb, as previously shown for several other Streptomyces species. By hybridization, it could be shown that the biosynthetic and resistance genes for pristinamycins I and pristinamycins II, except for the multidrug resistance gene ptr, are interspersed and seem to be organized as a single large cluster, covering less than 200 kb corresponding to 2.6% of the total size of the chromosome. The consequences and significance of such a genetic organization are discussed.

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey.

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.

Variable Number Tandem Repeat (VNTR) analysis reveals genetic diversity within Mycoplasma mycoides mycoides small colony isolates from Nigeria.

A Variable Number Tandem Repeat (VNTR) analysis was conducted on thirteen (13) M. mycoides mycoides Small Colony isolates from Nigeria using Tandem Repeat (TR) 34 which is a predicted lipoprotein located within the hypothetical protein MAG6170. The analysis revealed diversity within the M. mycoides mycoides Small Colony isolates with five different VNTR types indicated. Some correlation was determined between the VNTR types and their geographical origin. VNTR analysis may represent a useful, rapid first-line test for use in molecular epidemiological analysis of M. mycoides mycoides Small Colony for possible outbreak tracing and disease control.

Stress effect of different temperatures and air exposure during transport on physiological profiles in the American lobster Homarus americanus.

Homarus americanus is an important commercial species that can survive 2-3 days out of water if kept cool and humid. Once caught for commercial purpose and shipped around the world, a lobster is likely to be subjected to a number of stressors, including emersion and air exposure, hypoxia, temperature changes and handling. This study focused on the effect of transport stress and specifically at different animal body temperature (6 and 15 degrees C) and air exposure during commercial transport and recovery process in water. Animals were monitored, by hemolymph bleeding, at different times: 0 h (arrival time at plant) 3 h, 12 h, 24 h and 96 h after immersion in the stocking tank with a water temperature of 6.5+/-1.5 degrees C. We analysed the effects by testing some physiological variables of the hemolymph: glucose, cHH, lactate, total protein, cholesterol, triglycerides, chloride and calcium concentration, pH and density. All these variables appeared to be influenced negatively by high temperature both in average of alteration from the physiological value and in recovering time. Blood glucose, lactate, total protein, cholesterol were significantly higher in the group with high body temperature compared to those with low temperature until 96 h after immersion in the recovery tank.

A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

Heavy metal toxicity and differential effects on the hyperglycemic stress response in the shrimp Palaemon elegans.

Agricultural and industrial activities cause heavy metal pollution of the aquatic environment. The sensitivity of crustaceans to heavy metals is well documented. However, the hormonal and metabolic target of physiological functions affected by sublethal toxicity and stress responses have been scarcely investigated. Exposure of Palaemon elegans to increasing concentrations of heavy metals dissolved in artificial sea water resulted in an order of toxicity tested by LC(50) for 96 h in intact and eyestalkless animals in which Hg is the most toxic, followed by Cd, Cu, Zn, and Pb. Eyestalkless animals were found to be more sensitive than intact individuals. Heavy metals affect the blood glucose levels, yet manipulative stress does not. The intermediate sublethal concentrations of Hg, Cd, and Pb produced significant hyperglycemic responses within 3 h, while the highest concentrations elicited no hyperglycemia in 24 h. In contrast, animals exposed to Cu and Zn showed hyperglycemia even at high concentrations. This difference in response between Cu or Zn and the nonessential heavy metals Cd, Hg, or Pb can probably be explained by the physiological roles of the former in crustaceans and by tolerance adaptations. Involvement of the crustacean hyperglycemic hormone (cHH) was tested by routine bioassay on eyestalkless individuals; each group was injected with a two-eyestalk-equivalent extract from control animals or from shrimp exposed to high concentrations of Cd, Hg, Pb, or low concentrations of Cu or Zn. All showed a hyperglycemic response within 2 h. In contrast, extracts of eyestalk removed from animals that had developed a full hyperglycemic reaction after exposure to low concentrations of Hg, Cd, Pb, or high concentrations of Cu and Zn were depleted of cHH as shown by the attenuation of the response after injection of the extracts into eyestalkless animals. This generalized and predictable sublethal response can be used as a quantitative physiological biomarker for water quality monitoring assessment.

Lipopolysaccharide-induced hyperglycemia is mediated by CHH release in crustaceans.

Septicemia in crustaceans may occur occasionally due to Gram-negative opportunistic bacteria, especially under conditions of intensive aquaculture. The lipopolysaccharide (LPS) endotoxin induces in mammals septic shock and the activation by LPS of hormone release through the hypothalamo-pituitary axis is well known. In crustaceans an increase in circulating Crustacean hyperglycemic hormone and hyperglycemia are reported to result from exposure to several environmental stressors but the metabolic and hormonal effects of LPS in vivo are undescribed. A sublethal dose of LPS (Sigma, Escherichia coli 0111:B4) was injected into at least five individuals of species representative of crustacean taxa and life habits: Squilla mantis (Stomatopoda); the Decapoda Crangon crangon and Palaemon elegans (Caridea), Nephrops norvegicus (Astacidea), Munida rugosa and Paguristes oculatus (Anomura), Pilumnus hirtellus, Macropipus vernalis, Parthenope massena, and Ilia nucleus (Brachyura). Within 3 hr an increase in blood sugar developed ranging from 26.00 +/- 8.37 sd mg/dl in M. rugosa to 201.50 +/- 95. 91 sd mg/dl in P. oculatus and a significant increase of 79% in M. rugosa up to 1300% in P. hirtellus over control levels was observed. The involvement of eyestalk hormones in this generalized response was tested on S. mantis, M. vernalis, and P. elegans; LPS injected into eyestalkless animals did not elicit a significant hyperglycemic response compared with saline-injected controls. Eyestalkless animals injected with one eyestalk equivalent homogenate in saline from untreated animals did show a change in color from red to normal likely due to red pigment concentrating hormone and a hyperglycemic response within 2 hr. Eyestalkless animals injected with homogenate from LPS-treated shrimps showed the change in color but not the hyperglycemic response. It is concluded that LPS directly, or cytokines circulated upon challenge by the endotoxin, may act on the medulla terminalis X-organ-sinus gland complex and release CHH selectively eliciting an hyperglycemic stress response, after which CHH stores become relatively depleted.

A comparison of sagittal and vertical effects between bonded rapid and slow maxillary expansion procedures.

The purpose of this study was to determine the vertical and sagittal effects of bonded rapid maxillary expansion (RME), and bonded slow maxillary expansion (SME) procedures, and to compare these effects between the groups. Subjects with maxillary bilateral crossbites were selected and two treatment groups with 12 patients in each were constructed. The Hyrax screw in the RME treatment group and the spring of the Minne-Expander in the SME treatment group were embedded in the posterior bite planes, which had a thickness of 1 mm. At the end of active treatment these appliances were worn for retention for an additional 3 months. Lateral cephalometric radiographs were taken at the beginning and end of treatment, and at the end of the retention period. The maxilla showed anterior displacement in both groups. The mandible significantly rotated downward and backward only in the RME group. The inter-incisal angle and overjet increased in both groups. No significant differences were observed for the net changes between the two groups.

Comparison of dental arch and arch perimeter changes between bonded rapid and slow maxillary expansion procedures.

The purpose of this study was to evaluate and compare the dental effects of bonded rapid maxillary expansion (RME) and bonded slow maxillary expansion (SME) treatment methods. Subjects with a maxillary bilateral crossbite were selected and two treatment groups each with 12 patients were constructed. At the beginning of treatment, the average chronological ages were 11.96 years for the RME group and 12.31 years for the SME group. The Hyrax screw in the RME treatment group and the spring of the Minne-Expander in the SME treatment group were embedded in the posterior bite planes, which had a thickness of 1 mm. The treatment time for the RME group varied from 0.70 to 1.60 months and for the SME group 1.00-5.16 months. At the end of active treatment the appliances were worn for retentive purpose for an additional 3 months. Orthodontic casts taken at the beginning and end of treatment, and at the end of the retention period formed the material for the study. Increases in the transversal width between the upper molars, upper first premolars, upper canines, lower canines, and in the upper arch perimeter were obtained. The increase in the upper inter-canine width was found to be significantly greater in the RME group compared with the SME group. Regression analysis indicated that arch perimeter gain through the treatment could be predicted as 0.65 times the amount of the posterior expansion for the RME group and 0.60 times the amount of posterior expansion for the SME group.

Different bacterial lipopolysaccharides as toxicants and stressors in the shrimp Palaemon elegans.

This study compares the in vivo haemocytic response of shrimp, Palaemon elegans (Rathke) to different types of LPS injection. In particular it investigates to what degree and speed the haemocytopenia varies between LPSs from different sources. It further compares the tolerated doses of different LPSs in these animals and finds substantial differences in the various toxicity types. The work then relates this to blood glucose levels and stress-linked variations in glycaemic status. The order of LPS decreasing toxicity determined by LD50 at 96 h was: Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa 10, Escherichia coli K-235 and E. coli 0111:B4. Eyestalkless animals were more sensitive to LPS. The effects of injected LPS on circulating total blood cell count (THC) was tested. The results show that LPS caused a decrease in THC 8 h after injection and then the THC returned to the initial level and this effect depended on the LPS tested. E. coli K-235 was the most effective in causing haemocytopenia followed by E. coli 0111:B4, S. enteritidis, S. marcescens, and P. aeruginosa 10. Moreover, LPS-induced increases in the blood glucose level and the time and dose related curves of response obtained depended on the type of LPS tested. E. coli K-235 LPS was again the most effective in elevating blood glucose followed by E. coli 0111:B4, S. marcescens, S. enteritidis and then P. aeruginosa 10. No significant hyperglycaemia was induced in eyestalkless animals. An inverse order relationship between toxicity (LD50) and stress responses (hyperglycaemia and THC decrease) may suggest a defensive and adaptive role of the latter in occasional septicaemia.

Specific PCR identification of the T1 vaccine strains for contagious bovine pleuropneumonia.

A specific PCR test for the identification of the vaccine strains T1, T1/44 and T1sr, was developed. This PCR reaction is based on variations of DNA sequences in a region flanking one IS1296 copy. The specific primer pair MmmSCP1-T1M2 amplifies a 700-bp long DNA fragment in the T1 vaccine strains and gives no amplification with the 60 other Mycoplasma mycoides subsp. mycoides SC strains tested. This PCR will permit to distinguish the T1 strain from all other vaccine strains and therefore avoid possible confusions. In addition, it should enable better investigations of post-vaccinal reactions.

An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea.

The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-L-phenylalanine precursor of pristinamycin I.

Four pap genes (papA, papB, papC, papM) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI- phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.