A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

Effects of a synovial fluid substitute on early recovery after arthroscopic subacromial decompression of the shoulder.

PURPOSE: The purpose of this pilot study was to determine whether the use of a synovial fluid substitute (Viscoseal) after arthroscopic subacromial decompression (ASD) of the shoulder was safe (primary outcome) and effective in reducing the postsurgical pain on the day of surgery and the time from surgery to discharge (secondary outcomes), compared with patients undergoing standard ASD alone. METHODS: Forty-six patients with primary isolated shoulder subacromial impingement were randomly assigned to either undergo SAD alone (control group: n = 21) or to receive 10 ml Viscoseal into the subacromial space at the end of the procedure (treatment group: n = 25). RESULTS: No adverse events were reported in either group. All clinical scores improved significantly in each group from preoperative to 12-week follow-up (p < 0.01). The Viscoseal group experienced significantly (p = 0.001) less severe pain 4 h after the surgery {mean 54.0 ± 43.1, median 50 [interquartile range (IQR) 0-100]} and shorter time from surgery to discharge [mean 5.2 ± 1.4, median 5 (IQR 4-6)] than the control group [mean 102.4 ± 40.2, median 100 (IQR 50-150) and mean 11.0 ± 5.3, median 12 (IQR 6-16), respectively]. The Viscoseal group also required less analgesia postoperatively than the control group in the first 8 h: 24% of the Viscoseal required no analgesia, while all patients in the control group required analgesia; 24% of the control group required opiates compared with 4% in the Viscoseal group. CONCLUSION: Viscoseal was safe and well tolerated after shoulder arthroscopy. It provided excellent pain relief and a faster discharge time after ASD of the shoulder. The use of Viscoseal should be investigated in larger randomized controlled trials and for other shoulder arthroscopy procedures. LEVEL OF EVIDENCE: Level II, Pilot Prospective Comparative Study.

Demonstration that the BchH protein of Rhodobacter capsulatus activates S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase.

The bchH gene of Rhodobacter capsulatus has been cloned into an expression strain of Escherichia coli. Following induction of expression of the BchH protein, it was found that the E. coli strain also accumulated porphyrins with the fluorescence properties of protoporphyrin and zinc protoporphyrin. It was also found that the soluble BchH protein increased the activity of S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase, when mixed with membranes of an expression strain of E. coli into which the bchM gene (which encodes the methyltransferase) had been cloned, as well as membranes of a bchH mutant of R. capsulatus.

In vitro expression and activity of lycopene cyclase and beta-carotene hydroxylase from Erwinia herbicola.

The cyclisation of lycopene to beta-carotene and the hydroxylation of beta-carotene to zeaxanthin are common enzymatic steps in the biosynthesis of carotenoids in a wide range of bacteria, fungi, and plants. We have individually expressed in E. coli the two genes coding for these enzymatic steps in Erwinia herbicola. The cyclase and hydroxylase enzymes have apparent molecular weights of 43 kDa and 22 kDa, respectively, as determined by SDS-PAGE. Hydroxylase in vitro activity was obtained only in the cytoplasmic fraction. Cyclase also demonstrated enzyme activity in a crude cell-free lysate, although to a lesser extent.

Protein kinase C and adaptation to ethanol.

Adaptation to chronic ethanol exposure results in a decrease in sensitivity to the intoxicating effects of ethanol. Recent evidence indicates that changes in the expression and function of certain proteins involved in signal transduction are important for adaptation to ethanol. Using the neural cell line PC12, we found that chronic exposure to ethanol increases the expression and function of L-type voltage-gated calcium channels and enhances neural differentiation induced by nerve growth factor. Both of these responses to ethanol require protein kinase C (PKC). Chronic ethanol exposure activates PKC-mediated phosphorylation, in part, by increasing the expression of two PKC isozymes, delta and epsilon. The PKC family of enzymes may be important targets for the development of drugs that could modify adaptive and toxic consequences of chronic ethanol exposure.

Carotenoids of Erwinia herbicola and an Escherichia coli HB101 strain carrying the Erwinia herbicola carotenoid gene cluster.

Carotenoid pigments of Erwinia herbicola and a transformed strain of Escherichia coli carrying the carotenoid biosynthesis gene cluster of E. herbicola have been analyzed. Both organisms are capable of making essentially the same carotenoids, indicating that all of the genes required for the biosynthesis of the wild type E. herbicola carotenoids have been transformed intact into E. coli. The major products in both species of bacteria are beta-cryptoxanthin glucoside, zeaxanthin monoglucoside and zeaxanthin diglucoside. These compounds are the first example of secondary, non-allylic carotenoid glucosides. The absolute configuration 3R,3'R for zeaxanthin diglucoside was determined from its circular dichroism spectrum. Both species of bacteria also accumulate small amounts of hydrocarbon carotenes with similar cis/trans isomerization states.

Use of the membrane-impermeable guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate to demonstrate the orientation of light-harvesting proteins in Rhodobacter sphaeroides.

The radiolabeled guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate reacts with the epsilon-amino groups of accessible lysyl residues of membrane proteins under relatively mild labeling conditions, yielding labeled homoarginyl residues. Model studies have shown that the resulting homoarginyl residues do act as new cleavage sites for trypsin, but only at a very slow rate of hydrolysis. The reagent has been shown to be impermeable to the intracytoplasmic membranes of Rhodobacter sphaeroides: when cytoplasmic-side-out chromatophores were treated with the reagent, it reacted with all four of the light-harvesting proteins, all of which have one or more lysyl residues on the N-terminal sides of their hydrophobic regions. However, when periplasmic-side-out vesicles, prepared by cytochrome c affinity chromatography, were treated with the guanidinating reagent, three of the light-harvesting proteins (B850 alpha, B850 beta, and B870 beta) were not labeled. The only light-harvesting protein to be labeled (B870 alpha) was the only one of the four to have a lysyl residue on the C-terminal side of its hydrophobic region. Guanidinated B870 alpha polypeptides from both the cytoplasmic-side-out chromatophores and the periplasmic-side-out membrane vesicles were purified and digested with trypsin. The resulting peptide fragments were then separated by high-performance liquid chromatography and analyzed for radioactivity. The results have confirmed the asymmetric orientation of the light-harvesting proteins of R. sphaeroides, with their N-termini on the cytoplasmic side of the intracytoplasmic membrane. In the case of the B870 alpha subunit, the protein has been shown to be transmembrane with its C-terminus on the periplasmic side of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol enhances growth factor activation of mitogen-activated protein kinases by a protein kinase C-dependent mechanism.

Excessive alcohol consumption alters neuronal growth and causes striking elongation of axons and dendrites in several brain regions. This could result from increased sensitivity to neurotrophic factors, since ethanol markedly enhances nerve growth factor (NGF)- and basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth in the neural cell line PC12. The mechanism by which ethanol enhances growth factor responses was investigated by examining activation of mitogen-activated protein kinases (MAP kinases), a key event in growth factor signaling. Ethanol (100 mM) increased NGF- and bFGF-induced activation of MAP kinases. This increase, like ethanol-induced increases in neurite outgrowth, was prevented by down regulation of beta, delta, and epsilon protein kinase C (PKC) isozymes. Since chronic ethanol exposure specifically upregulates delta and epsilon PKC, these findings suggest that ethanol promotes neurite growth by enhancing growth factor signal transduction through a delta or epsilon PKC-regulated pathway.

Overexpression of epsilon-protein kinase C enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and neurite outgrowth.

Protein kinase C (PKC) activation enhances neurite outgrowth in several cell lines and primary neurons. The PKC isozymes that mediate this response are unknown. One clue to their identity has come from studies using PC12 cells treated with ethanol. In these cells, ethanol increases levels of delta-PKC and epsilon-PKC and markedly enhances nerve growth factor (NGF)-induced neurite outgrowth and activation of mitogen-activated protein (MAP) kinases by a PKC-dependent mechanism. Since these findings suggest that delta-PKC or epsilon-PKC can promote neural differentiation, we studied neurite outgrowth in stably transfected PC12 cell lines that overexpress these isozymes. Overexpression of epsilon-PKC markedly increased NGF-induced neurite outgrowth. This effect was blocked by down-regulating PKC or by treating cells with the PKC inhibitor GF 109203X. In addition, overexpression of epsilon-PKC enhanced NGF-induced phosphorylation of MAP kinases. In contrast, overexpression of delta-PKC did not alter responses to NGF. These results demonstrate that epsilon-PKC promotes NGF-induced neurite outgrowth by enhancing NGF signal transduction. These findings suggest a role for epsilon-PKC in neural differentiation and plasticity.

Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed uridine diphosphate binding site.

Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono- and diglucosides of zeaxanthin. We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of zeaxanthin. The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass. In vitro enzymatic activity was demonstrated using UDP-[14C]glucose and zeaxanthin as substrates. The product zeaxanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography. The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-37 degrees C, respectively. A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated. The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl- and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates. Based on these similarities, we propose that this conserved sequence is part of the UDP binding site.

An inhibitory fragment derived from protein kinase Cepsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters.

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli.

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, beta-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for beta-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.