A new enzyme superfamily - the phosphopantetheinyl transferases

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.     

Comparison of the genomic short regions of a vaccine strain (SA-2) and a virulent strain (CSW-1) of infectious laryngotracheitis virus (Gallid herpesvirus 1)

Restriction enzyme linkage maps were produced for the genomic short region of the virulent infectious laryngotracheitis virus (CSW-1 strain). After comparison with the equivalent restriction enzyme linkage maps for the infectious laryngotracheitis virus SA-2 strain (a vaccine strain), it was determined that the maps for the short regions of the two strains were identical, apart from a single section in each of the inverted terminal repeats. Each inverted terminal repeat of the SA-2 strain was discovered to contain 467 base pairs more DNA than the CSW-1 strain's inverted terminal repeats. This extra DNA was more precisely mapped entirely within the EcoRI fragments D and d of SA-2, which were found to form part of the SmaI fragments U and P of SA-2 and Q and b of SA-2 and to contain one SmaI restriction enzyme site.     

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.     

Lidocaine-induced CNS toxicity--a case report

One hundred and twenty-six cases of cerebellopontine angle tumors with various histologies are presented. Results of 75 operated vestibular neurinomas, 22 meningiomas, and 16 tumors with other histologies are discussed. The method of irradiation and non-radical surgery may be an alternative for treatment.     

Multiple members of the leucokinin neuropeptide family are present in cerebral and abdominal neurohemal organs in the cockroach Leucophaea maderae

Two neurohemal organs of the cockroach Leucophaea maderae, the corpora cardiaca and the lateral heart nerve are known to contain leucokinin immunoreactive material. We examined the corpora cardiaca and the lateral heart nerve to establish whether these neurohemal organs store all 8 known leucokinin isoforms or if the leucokinins have a differential distribution. Extracts of corpora cardiaca and abdominal hearts with attached lateral heart nerve were separated on reversed phase high performance liquid chromatography (rpHPLC), then tested for leucokinin immunoreactivity by a radioimmunoassay (RIA) able to detect all 8 leucokinin isoforms. Extracts from brain and optic lobes were also separated and assayed in the RIA. Synthetic leucokinin 1-8 were subjected to rpHPLC and their different retention times established by RIA for reference. Leucokinin immunoreactive material originating from the corpora cardiaca and lateral heart nerves eluted in fractions corresponding to those of the synthetic leucokinin 1-8. In this study we have thus demonstrated that probably all 8 leucokinin isoforms are stored in the corpora cardiaca and the lateral heart nerve. These observations suggest that all 8 leucokinins are likely to be released as neurohormones into the circulation.     

PAX genes and human neural tube defects: an amino acid substitution in PAX1 in a patient with spina bifida

From studies in the mouse and from the clinical and molecular analysis of patients with type 1 Waardenburg syndrome, particular members of the PAX gene family are suspected factors in the aetiology of human neural tube defects (NTD). To investigate the role of PAX1, PAX3, PAX7, and PAX9, allelic association studies were performed in 79 sporadic and 38 familial NTD patients from the Dutch population. Sequence variation was studied by SSC analysis of the paired domain regions of the PAX1, PAX7, and PAX9 genes and of the complete PAX3 gene. In one patient with spina bifida, a mutation in the PAX1 gene was detected changing the conserved amino acid Gln to His at position 42 in the paired domain of the protein. The mutation was inherited through the maternal line from the unaffected grandmother and was not detected in 300 controls. In the PAX3 gene, variation was detected at several sites including a Thr/Lys amino acid substitution in exon 6. All alleles were present among patients and controls in about the same frequencies. However, an increased frequency of the rare allele of a silent polymorphism in exon 2 was found in NTD patients, but no significant association was observed (p = 0.06). No sequence variation was observed in the paired domain of the PAX7 and PAX9 genes. Our findings so far do not support a major role of the PAX genes examined in the aetiology of NTD. However, the detection of a mutation in PAX1 suggests that, in principle, this gene can act as a risk factor for human NTD.     

Interaction of nonylphenol and hepatic CYP1A in rats

Both estradiol and nonylphenol (NP) inhibited hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of beta-naphthoflavone-treated rats. Enzyme kinetic analyses (Lineweaver-Burk plots) using different estradiol and NP concentrations with graded increases in the concentrations of the substrate, ethoxyresorufin, showed that the inhibition was of a competitive nature at all concentrations of estradiol or NP used. Thus, the mechanism by which NP inhibits EROD activity is similar to that of estradiol. NP, however, was much less potent than estradiol. Young rats treated in vivo with 80 mg/kg body weight of NP demonstrated a slight but significant decrease in their hepatic microsomal EROD activity and CYP1A protein as measured by western blot analysis. In addition, treatment with NP led to a decrease in the steady-state levels of hepatic CYP1A mRNA in rats, suggesting that NP acted at the pre-translational level. The competitive nature of inhibition by NP on hepatic microsomal EROD activity indirectly suggests that this compound is a possible substrate of the CYP1A enzyme. Furthermore, NP had a moderate modulating effect on the expression of CYP1A in rat liver.     

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization

Human and animal studies have clearly demonstrated the advantageous effects of sensorially enriched rearing environments. Nevertheless, little work has been done concerning the long-lasting persistence of all these behavioral modifications. To undertake this question, a very early enrichment animal model was used. From days 10 to 24 after birth, 28 male albino rats were exposed to a multisensory stimulated environment, while other 28 littermates constituted the control group. At 3 and 6 months old two cognitive abilities were analyzed; the spatial working memory (short term memory) and the latent learning capacity (long term memory). The results evidenced an improved working memory in both 3 and 6 months old rats exposed to the early enriched environment. Moreover, the adult early stimulated group performed as well as younger subjects both on error scores and speed to solve this test. Only in the adult group of rats a superior latent learning capacity of stimulated subjects was evidenced. To conclude, the early enriched environment induced: a) persistent cognitive benefits in the adult rat and b) a more relevant influence on the subsequent behavior of older rather than younger subjects.     

Variability of clinical and pressure-flow study variables after 6 months of watchful waiting in patients with lower urinary tract symptoms and benign prostatic enlargement

PURPOSE: We quantified the physiological variability of clinical and pressure-flow study variables in patients with symptomatic benign prostatic enlargement. MATERIALS AND METHODS: Symptom scores were measured, and advanced urodynamic studies with pressure-flow analysis were performed in 178 patients before and 6 months after a period a watchful waiting. RESULTS: Patients without bladder outlet obstruction experienced significant symptomatic improvement. Symptoms in patients with obvious bladder outlet obstruction did not improve significantly. The reproducibility of mean pressure-flow variables was evident. However, there was an important intra-individual variability. Patients with obvious bladder outlet obstruction showed a significant decreases in detrusor pressure at maximal flow of 14cm. water, a significant decrease in the urethral resistance factor of 7 cm. water and a significant decrease of 1 obstruction class on the linear passive urethral resistance relation nomogram, indicating less severe bladder outlet obstruction. CONCLUSIONS: Mean differences among therapy groups must be regarded critically, especially when the difference are slight and possibly within physiological variability.     

The effect of isradipine administration on existing fatty streaks in the cholesterol-fed rabbit: a morphometric study

Previous studies have shown that the administration of certain calcium channel blocking drugs (at an appropriate time point) can reduce the severity of atherosclerotic lesion formation. This study was undertaken to determine if the administration of isradipine would reverse established lesions produced by feeding rabbits an atherogenic diet. Rabbits were fed cholesterol for three weeks and examined directly, or after being left for a four week washout period, with or without a daily oral supplement of isradipine. Fatty streaks were well established after three weeks of cholesterol feeding and were more extensive at the end of the washout period, as indicated by gross changes in the volume of the intima per unit length of aorta. When isradipine was administered during the washout period, the volume of the intima per unit length of aorta fell to levels below those produced by cholesterol feeding for three weeks alone. The major components of the lesions affected to accommodate these changes were the foam cells and myointimal cells; these were examined in detail using morphometry and lipid cytochemistry. The mean volume of intima/cm of aorta occupied by foam cells and myointimal cells both fell by more than 60% to levels lower than those found after three weeks of cholesterol feeding alone. The volume of the extracellular space of the intima occupied by cytochemically demonstrable unesterified and esterified cholesterol was reduced by isradipine administration as was that of foam cells, all to levels lower than those found after three weeks of cholesterol feeding alone. These data indicate that the administration of isradipine during a washout period, after cholesterol feeding, can promote the regression of fatty streak lesions.