Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase.
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BACKGROUND: Polyketides are important compounds with antibiotic and anticancer activities. Several modular polyketide synthases (PKSs) contain a terminal thioesterase (TE) domain probably responsible for the release and concomitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PKSs, its mechanism and specificity are of considerable interest. RESULTS: The TE domain of the 6-deoxyerythronolide B synthase was overexpressed in Escherichia coli. When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant hydrolytic specificity towards substrates that mimic important features of its natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the terminal PKS module (by as much as 100-fold compared with separate TE and PKS 'domains'). CONCLUSIONS: The inability of the TE domain alone to catalyze cyclization suggests that macrocycle formation results from the combined action of the TE domain and a PKS module. The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesis of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both terminal and pre-terminal modules, which may explain the ability of certain naturally occurring PKSs, such as the picromycin synthase, to generate both 12-membered and 14-membered macrolide antibiotics. (+info)
Morphology of intraepithelial corpuscular nerve endings in the nasal respiratory mucosa of the dog.
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Corpuscular nerve endings in the nasal respiratory mucosa of the dog were investigated by immunohistochemical staining specific for protein gene product 9.5 by light and electron microscopy. In the nasal respiratory mucosa, complex corpuscular endings, which displayed bulbous, laminar and varicose expansions, were distributed on the dorsal elevated part of the nasal septum and on the dorsal nasal concha. The endings were 300-500 microm long and 100-250 microm wide. Some axons gave rise to a single ending while others branched into 2 endings. Cryostat sections revealed that the corpuscular endings were located within the nasal respiratory epithelium. On electron microscopy, immunoreactive nerve terminals that contained organelles, including mitochondria and neurofilaments, were observed within the epithelial layer near the lumen of the nasal cavity. Some terminals contacted the goblet cell. Such terminal regions were covered by the cytoplasmic process of ciliated cells and were never exposed to the lumen of the nasal cavity. These nerve endings are probably activated by pressure changes. (+info)
Depletion of cutaneous peptidergic innervation in HIV-associated xerosis.
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Severe xerosis occurs in approximately 20% of human immunodeficiency virus seropositive patients. Changes in cutaneous innervation have been found in various inflammatory skin diseases and in xerotic skin in familial amyloid. We have therefore carried out a quantitative examination of the cutaneous peptidergic innervation in human immunodeficiency virus-associated xerosis. Immunohistochemistry and image analysis quantitation were used to compare total cutaneous innervation (protein gene product 9.5), calcitonin gene-related peptide, substance P, and vasoactive intestinal peptide peptidergic fibers, at two sites in the skin of human immunodeficiency virus-associated xerosis patients (upper arm, n = 12; upper leg, n = 11) and site-matched seronegative controls (upper arm, n = 10; upper leg, n = 10). Measurement of lengths of fibers of each type was carried out for each subject in the epidermis and papillary dermis, and around the sweat glands. Immunostained mast cells in these areas were counted. Epidermal integrity and maturation were assessed by immunostaining for involucrin. There were significant (Mann-Whitney U test; p < 0.02) decreases in total lengths of protein gene product 9.5 fibers in both epidermis/papillary dermis and sweat gland fields; of calcitonin gene-related peptide innervation in the epidermis/papillary dermis; and of substance P innervation of the sweat glands. There were no differences in the distribution of mast cells, or in the epidermal expression of involucrin. Depletion of the calcitonin gene-related peptide innervation may affect the nutrient blood supply of the upper dermis, and the integrity and function of basal epidermis and Langerhans cells. Diminished substance P innervation of the sweat glands may affect their secretory activity. Both of these changes may be implicated in the development of xerosis. (+info)
The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat.
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1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids. (+info)
Identification of peroxisomal acyl-CoA thioesterases in yeast and humans.
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A computer-based screen of the Saccharomyces cerevisiae genome identified YJR019C as a candidate oleate-induced gene. YJR019C mRNA levels were increased significantly during growth on fatty acids, suggesting that it may play a role in fatty acid metabolism. The YJR019C product is highly similar to tesB, a bacterial acyl-CoA thioesterase, and carries a tripeptide sequence, alanine-lysine-phenylalanineCOOH, that closely resembles the consensus sequence for type-1 peroxisomal targeting signals. YJR019C directed green fluorescence protein to peroxisomes, and biochemical studies revealed that YJR019C is an abundant component of purified yeast peroxisomes. Disruption of the YJR019C gene caused a significant decrease in total cellular thioesterase activity, and recombinant YJR019C was found to exhibit intrinsic acyl-CoA thioesterase activity of 6 units/mg. YJR019C also shared significant sequence similarity with hTE, a human thioesterase that was previously identified because of its interaction with human immunodeficiency virus-Nef in the yeast two-hybrid assay. We report here that hTE is also a peroxisomal protein, demonstrating that thioesterase activity is a conserved feature of peroxisomes. We propose that YJR019C and hTE be renamed as yeast and human PTE1 to reflect the fact that they encode peroxisomal thioesterases. The physical segregation of yeast and human PTE1 from the cytosolic fatty acid synthase suggests that these enzymes are unlikely to play a role in formation of fatty acids. Instead, the observation that PTE1 contributes to growth on fatty acids implicates this thioesterase in fatty acid oxidation. (+info)
Morphological changes in periodontal mechanoreceptors of mouse maxillary incisors after the experimental induction of anterior crossbite: a light and electron microscopic observation using immunohistochemistry for PGP 9.5.
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Ruffini nerve endings (mechanoreceptors) in the periodontal ligament (PDL) of mouse incisors were examined to elucidate whether experimentally-induced crossbites cause any changes or abnormalities in their morphology and distribution. Anterior guiding planes were attached to the mandibular incisors of 3-week-old C3H/HeSlc mice. At 3 days and 1, 2, 4, 6, and 8 weeks post-attachment of the appliance, the mice were sacrificed by perfusion fixation. Frozen sagittal cryostat sections of the decalcified maxillary incisors were processed for immunohistochemistry of protein gene product 9.5, followed by histochemical determination of tartrate-resistant acid phosphatase activity to reveal sites of alveolar bone resorption. Despite the absence of bone resorption within the lingual PDL of control mice, distinct resorption sites were seen in the respective regions of the experimental animals. Unlike the controls, many Ruffini endings showing vague and swollen contours, with unusually long and pedunculated micro-projections were observed in the affected lingual PDL of the incisors in the experimental animals with short-term anterior crossbite induction. Club-shaped nerve terminations with few, if any, micro-projections were observed in the lingual PDL of experimental animals with long-term induction, as well as in aged control mouse incisors. Differences in the distribution of Ruffini endings were also observed. These results indicate that changing the direction of the force applied to the PDL results in rapid and prolonged changes in the morphology of Ruffini-like mechanoreceptors. (+info)
Development of the chick olfactory nerve.
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Gonadotropin releasing hormone (GnRH) is produced and secreted by neurons dispersed throughout the septal-preoptic and anterior hypothalamic areas in adult birds and mammals. These neurons, essential for a functional brain-pituitary-gonadal axis, differentiate in the olfactory placode, the superior aspect of which forms the olfactory epithelium. To reach their final placement within the brain, GnRH neurons migrate out of the epithelium and along the olfactory nerve to the CNS. This nerve is essential for the entrance of GnRH neurons into the CNS. Due to the importance of the nerve for the proper migration of these neurons, we have used immunocytochemistry, DiI labeling and 1 microm serial plastic-embedded sections to characterize the nerve's earliest development in the embryonic chick (stages 17-21). Initially (stage 17) the zone between the placode and prosencephalon is a cellular mass contiguous with the placode. This cluster, known as epithelioid cells, is positive for some but not all neuronal markers studied. The epithelium itself is negative for all neuronal and glial markers at this early stage. By stage 18, the first neurites emerge from the epithelium; this was confirmed at stage 19 by examination of serial 1 microm plastic sections. There is sequential acquisition of immunoreactivity to neuronal markers from stage 18 to 21. The glial component of the nerve appears at stage 21. Axons originating from epithelium, extend to the border of the CNS as confirmed by DiI labeling at stage 21. Small fascicles have entered the CNS at this stage. As previously reported, GnRH neurons begin their migration between stages 20-21 and have also arrived at the border of the brain at stage 21. Despite the penetration of neurites from the olfactory nerve into the CNS, GnRH neurons pause at the nerve-brain junction until stage 29 (2 1/2 days later) before entering the brain. Subsequent studies will examine the nature of the impediment to continued GnRH neuronal migration. (+info)
Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity.
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The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction. (+info)