Search PubMed⌕ Search

Biomedical subjects

G Schuler

Publications and source records attributed to G Schuler.

At least 181 records · Page 10Linked to original sources
PubMed1997

Dendritic cells as immunogens for human CTL responses.

The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses.

Blood Cells↗
PubMed1997

[Dermatology and the Internet--uses for the clinic and research].

The Internet is about to become the most important source of information in virtually any academic profession. Dermatology has a growing presence in many areas of the Internet: discussion groups and continuing education programs invite regular participation, dermatologic data in the form of text and images provide support in daily clinical and scientific work. In this paper important sources of dermatologically relevant information on the Internet are given, and the Dermatologic Online Image Atlas (DOIA) which is available on the World Wide Web is introduced.

Computer Communication Networks↗
PubMed1997

[Comparative studies of the rate of restenosis after implantation of Palmaz-Schatz and AVE-Micro stents].

Late results of interventional procedures utilizing coronary stents are largely determined by the rate of restenosis. So far few data are available addressing the effect of stent design, implantation pressure and morphologic factors on this crucial variable. Therefore we analyzed the coronary angiograms obtained in 259 patients before, immediately after and at 3 to 6 months following stent implantation for obstructive coronary disease. A total of 196 AVE-Micro-Stents and 142 Palmaz-Schatz-Stents were implanted into 307 stenoses. In 126 stenoses there were implanted only Palmaz-Schatz-Stents, in 170 only AVE-Micro-Stents and in 11 stenoses there were implanted Palmaz-Schatz- as well as Micro-Stents. Restenosis was defined as an over 50% stenosis at follow up. No significant difference was detected with regard to global restenosis rate at an average of 4 months following implantation (Palmaz-Schatz 33%, Micro-Stent 27%). If results were analyzed according to implantation pressure however, there was a significantly lower restenosis rate for AVE-Micro-Stents implanted with > 10 atm (17%) as compared to < or = 10 atm (35%, p < 0.02) and as compared to Palmaz-Schatz-Stents (34%, p < 0.02), which were also implanted with high pressure over 10 atm. In addition to implantation pressure, vessel segment and morphology of stenosis proved to be important determinants of late results. In this series of patients the AVE-Micro-Stent compared favourably to the Palmaz-Schatz-Stent not only with respect to a significantly lower restenosis rate, when implanted with pressures > 10 atm, but also with regard to its superior flexibility and handling characteristics.

Aged↗
PubMed1997

Resolution requirements for digital images in dermatology.

BACKGROUND: The digital image has become an important tool in dermatology because of the rapid development of computer hardware, networks, and the World Wide Web. OBJECTIVE: Our purpose was to examine the resolution requirements for digital images. METHODS: Slides of eight selected images showing characteristic lesions were produced in five different resolutions each, ranging from 192 x 128 x 24 (192 pixels x, 128 pixels y, and 24-bit color depth) up to 3072 x 2048 x 24. They were compared side by side by a group of six experienced dermatologists using a standardized questionnaire. RESULTS: Images at the resolution of 768 x 512 x 24 were perceived as equivalent to higher resolutions, whereas a clear difference was visible between 768 x 512 x 24 and 384 x 256 x 24. The slide projector had a significant influence on the rating of the images. CONCLUSION: For digital images in dermatology a resolution of 768 x 512 x 24 is suitable to recognize the relevant details of the source image.

Dermatology↗
PubMed1997

Intravenous immunoglobulin treatment in therapy-resistant epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita is an uncommon autoimmune bullous disease of the skin and mucous membranes. It is chronic, disabling, and difficult to treat. We describe a case of severe epidermolysis bullosa acquisita of 7 years' duration that had been treated with azathioprine, corticosteroids, chlorambucil, plasma exchanges, cyclophosphamide, cyclosporine, and colchicine without any lasting effect. Seven cycles of treatment were administered with immunoglobulin given intravenously at a low dose, 40 mg/kg body weight daily for 5 days. The patient was free of disease for 10 months after the initiation of therapy. We suggest that low-dose regimens of immunoglobulins may be as effective in this disease as the high-dose regimens suggested in the literature, and at much lower cost.

Adult↗
PubMed1997

Effects of endurance training on mitochondrial ultrastructure and fiber type distribution in skeletal muscle of patients with stable chronic heart failure.

OBJECTIVES: The present study was designed to evaluate the effects of an ambulatory training program in patients with chronic heart failure (CHF) on the ultrastructural morphology of mitochondria and fiber type distribution of skeletal muscle and its relation to peripheral perfusion. BACKGROUND: Recent studies in patients with CHF have suggested that intrinsic abnormalities in skeletal muscle can contribute to the development of early lactic acidosis and fatigue during exercise. METHODS; Patients were prospectively randomized to either a training group (n = 9; mean [+/- SD] left ventricular ejection fraction [LVEF] 26 +/- 10) participating in an ambulatory training program or to a physically inactive control group (n = 9; LVEF 28 +/- 10%). At baseline and after 6 months, patients underwent symptom-limited bicycle exercise testing with measurement of central and peripheral hemodynamic variables as well as percutaneous needle biopsies of the vastus lateralis muscle. The mitochondrial ultrastructure of skeletal muscle was analyzed by ultrastructural morphometry; cytochrome c oxidase activity was visualized by histochemistry and subsequently quantitated by morphometry. The fiber type distribution was determined by adenosine triphosphatase staining. RESULTS: After 6 months of exercise training there was a significant increase of 41% in the surface density of cytochrome c oxidase-positive mitochondria (SVMOcox+) (p < 0.05 vs. control) and of 43% in the surface density of mitochondrial cristae (SVMC) (p < 0.05 vs. control). Furthermore, exercise training induced a 92% increase in the surface density of the mitochondrial inner border membrane (p < 0.05 vs. control). In contrast, the total number of cytochrome c oxidase-positive mitochondria remained essentially unchanged. Exercise-induced improvement in peak oxygen uptake was closely linked to changes in SVMOcox+ (p < 0.01, r = 0.66). After exercise training, changes in submaximal femoral venous lactate levels were not related to changes in submaximal leg blood flow (r = -0.4), but were inversely related to changes in the volume density of mitochondria (p = 0.01; r = -0.6) as well as to changes in SVMC (p < 0.05; r = -0.5). After exercise training there was a "reshift" from type II to type I fibers (p < 0.05 vs. control). CONCLUSIONS: Patients with CHF who engage in regular physical exercise show enhanced oxidative enzyme activity in the working skeletal muscle and a concomitant reshift to type I fibers. These exercise-induced changes in oxidative capacity appear to be unrelated to changes in peripheral perfusion.

Electron Transport Complex IV↗
PubMed1997

Fetal villosity and microvasculature of the bovine placentome in the second half of gestation.

The architecture of the fetal villous tree and its vasculature in the bovine placentome were studied in the second half of gestation using both conventional histology and histology of ink-filled blood vessels. These were compared with corrosion casts of plastic fillings of the vasculature, prepared for scanning electron microscopy. This combination of morphological methods allows perception of the villous tree throughout gestation from broad-conical to tall-conical form where branch ramification occurs mainly at right angles to the stem. The stem villus typically contains a single central artery and several peripheral veins arranged in parallel. The proximal branches to the stem, the intermediate villi, contain a central arteriole and accompanying venules. The distal branches, the terminal villi, enclose capillary convolutions which consist of an afferent arterial capillary limb, capillary loops and efferent venous capillary limbs. Vascular interconnections exist within the terminal villi, as capillaries or venules between the capillary convolutions, serially bridging them in up to 5 places, and as capillary anastomoses between the capillary loops. Coiling and sinusoidal dilatations of these loops develop near the end of gestation. The intraplacentomal rearrangement of villous trees with progressive gestation and their morphological vascular adaptations are discussed in relation to placental function, including the ever increasing need for transplacental substance exchange. This adaptation allows the blood to traverse the shortest possible arterioarteriolar route to the periphery of the trees where exchange takes place. The need for an increasing blood flow stimulates capillary growth and at the same time optimises the blood flow reaching the placental barrier represented by the vessel cast surface. The capillaries also carry the blood back into the very voluminous system of venules and veins where back diffusion may occur. The total volume of terminal villi of bovine placentome, the 'working part' of villous trees, hence distinctly increases with respect to the stem and intermediate villi, the 'supplying part' of the villous tree. In morphological terms the efficiency of the bovine transplacental diffusional exchange is higher than in the closely related 'co-ruminants' sheep and goats and distinctly higher when compared with the human placenta.

Animals↗
PubMed1997

Determination of free and conjugated oestrogens in peripheral blood plasma, feces and urine of cattle throughout pregnancy.

In order to further characterize oestrogen production and metabolism during bovine pregnancy, free (f) and conjugated (c) estrone (E1), total free and conjugated oestrogens (tfcOe) and total free oestrogens (tfOe) were determined as marker oestrogens in blood plasma respectively in urine and feces of 10 pregnant cows. For the determination of individual oestrogens blood, urine and feces samples of days 240, 200, 160, 100, 60, 30, 10 and 5 prior to parturition were pooled and the free, sulfo (sc)- and glucuconjugated (gc) forms of E1, 17 beta-estradiol (E2 beta) and 17 alpha-estradiol (E2 alpha) were obtained following differential enzyme hydrolysis and separation by HPLC; hormone assay was by established RIA-procedures. FE1 and cE1 concentration in blood plasma, tfOe in feces and tfcOe in urine showed a similar pattern. A first rise occurred between days 110 and 120 of pregnancy, an additional overproportional rise commenced at around days 230-250. Highest concentrations were measured in feces (tfOe ca. 500 ng/g 1 day a. p.), followed by urine (tfcOe ca. 3.5 ng/mosmol 2 days a. p.) and blood plasma (fE1 ca. 8 nmol/l and cE1 ca. 20 nmol/l 2 days a. p.). Determination of individual oestrogens in blood plasma revealed that fE2 beta and fE2 alpha could only be found 10 days a. p. while the conjugated forms could already be detected on days 100 and 160 a. p. With 62% E1 was the dominant oestrogen, followed by E2 alpha (37%) and E2 beta (1.0%); E1 occurred predominantly as sulfate, E2 alpha and E2 beta predominantly as glucuronide. Main metabloite in feces was fE2 alpha (56.7%), followed by fE2 beta (32%) and fE1 (11.3%); conjugated oestrogens were not detected. Main metabolite in urine was scE1 followed by gcE2 alpha and gcE2 beta. ScE2 alpha and scE2 beta were not detected or were present in small quantities only. Hormonal changes over time were highly significant. Main product of placental oestrogen synthesis is scE1, the concentrations of f and c E2 beta and E2 alpha in plasma largely result from oestrogen metabolism and enterohepatic circulation.

Animals↗
PubMed1997

Dendritic cells: from ignored cells to major players in T-cell-mediated immunity.

Dendritic cells form a system of leukocytes specialized to stimulate resting T cells in vivo. Dendritic cells are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as 'nature's adjuvant'. The manifold specializations underlying this in vivo immunostimulatory function are becoming increasingly clear. Methods have been developed to generate large numbers of dendritic cells from hematopoietic precursors in vitro. These techniques now allow molecular studies as well as the use of antigen-charged dendritic cells to vaccinate patients against tumors (e.g. B-cell lymphoma or melanoma) and infection (e.g. HIV-1). Recent data suggest that besides the classical immunostimulatory dendritic cells which belong to the myeloid lineage, there exist regulatory dendritic cells related to the lymphoid lineage. These lymphoid-derived dendritic cells which at least in part express Fas-ligand appear to be involved in the induction of central as well as peripheral tolerance, and in the future might allow a novel approach to induce tolerance in transplantation, autoimmunity, and allergy.

Animals↗
PubMed1996

Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability.

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-alpha. The other method makes use of the more abundant CD34- precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day 'maturation culture' to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes ('veils'), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig. cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8-3.3 x 10(6) mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Cell Culture Techniques↗
PubMed1996

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood.

We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 ml of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.

Cell Culture Techniques↗
PubMed1996

High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

Animals↗
PubMed1996

Predictive value of lipid profile for salutary coronary angiographic changes in patients on a low-fat diet and physical exercise program.

In this study, 113 patients with modestly elevated levels of low-density lipoprotein cholesterol (<210 mg/dl) and coronary artery disease were randomized to an intervention group (n=56) or a control group (n=57). The intervention program consisted of daily exercise and a low-fat diet according to the American Heart Association's recommendation phase III; patients in the control group received "usual care" rendered by their private physician. After 1 year, complete data were available for all 92 patients (intervention: n=40; control: n=52) who underwent repeat coronary angiography. During the study course, patients in the intervention group showed an increase in apolipoprotein A-I(123 +/- 18 vs 129 +/- 20 mg/dl; p < 0.02) and apolipoprotein A-I/B (1.3 +/- 0.4 vs 1.5 +/- 0.4; p <0.01) and a decrease in apolipoprotein B (99 +/- 20 vs 89 +/- 18 mg/dl; p < 0.01), while apolipoprotein A-II remained unchanged (38 +/- 6 vs 38 +/- 6 mg/dl; p=NS). In the control group, there were no significant changes (apolipoprotein A-I, 124 +/- 17 vs 128 +/- 13 mg/dl; apolipoprotein A-II, 38 +/- 6 vs 39 +/- 6 mg/dl; apolipoprotein B, 100 +/- 21 vs 99 +/- 16 mg/dl; apolipoprotein A-I/B, 1.3 +/- 0.3 vs 1.4 +/- 0.5; all p=NS). As previously reported, there was a significant retardation of progression in patients in the intervention group (progression 23%, no change 45%, regression 32%) compared with the control group (progression 48%, no change 35%, regression 17%) (p < 0.05). Although retardation of progression was significantly associated with an increase in apolipoprotein A-I/B and a decrease in apolipoprotein B (p < 0.05), these gave way in multivariate analysis to changes in total cholesterol/high-density lipoprotein cholesterol, absolute levels of low-density lipoprotein cholesterol, and, in a subgroup of patients, to leisure-time physical activity (all p < 0.05). These data demonstrate that an intervention based on a low-fat diet and intensive physical exercise is capable of improving apolipoprotein levels, associated with retardation of progression of coronary artery disease. However, total cholesterol/high-density lipoprotein cholesterol and low-density lipoprotein cholesterol appear superior to apolipoproteins as metabolic markers for effective treatment in patients with coronary artery disease.

Adult↗
PubMed1996

An improved isolation method for murine migratory cutaneous dendritic cells.

Dendritic cells are highly specialized accessory cells for the initiation of primary immune responses. They occur as trace populations in non-lymphoid and lymphoid organs. Therefore, the isolation and enrichment of primary dendritic cells is difficult and time-consuming. This applies also to dendritic cells from skin, i.e. epidermal Langerhans cells and dermal dendritic cells. Recently introduced skin organ cultures serve as a convenient source for primary cutaneous dendritic cells. We report here a refinement of such cultures in which cutaneous dendritic cells emigrate spontaneously into the culture medium. Murine ear skin is cultured for a total of 3 days in three sequential 24 h steps. This simple modification doubles or triples the yields of dendritic cells that can be obtained and up to 30,000 dendritic cells can be recovered from one ear half. This represents 50-70% (range 30-80%) of all viable cells. The cells are mature dendritic cells that possess potent T cell stimulatory function. Compared to the classical methods of preparing epidermal Langerhans cells by trypsinization this technique is easier and quicker; it does not require enzymes such as trypsin, and it yields similar numbers of mature dendritic cells. It should prove useful for further studies of dendritic cells of the skin.

Animals↗
PubMed1996

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

Animals↗
PubMed1996

Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.

The capacity to migrate from peripheral tissues, where antigen is encountered, to lymphoid organs, where the primary immune response is initiated, is crucial to the immunogenic function of dendritic cells (DC). The skin is a suitable tissue to study migration. DC were observed to gather in distinct nonrandom arrays ("cords") in the dermis upon culture of murine whole skin explants. It is assumed that cords represent lymphatic vessels. Using a similar organ culture model with human split-thickness skin explants, we investigated migration pathways in human skin. We made the following observations. 1) Spontaneous emigration of Langerhans cells took place in skin cultured for 1-3 d. Nonrandom distribution patterns of strongly major histocompatibility complex class II-expressing DC (cords) occurred in cultured dermis. A variable, yet high (>50%) percentage of these DC coexpressed the Birbeck granule-associated antigen "Lag." Ultrastructurally, the cells corresponded to mature DC. 2) Electron microscopy proved that the dermal structures harboring the accumulations of DC (i.e., cords) were typical lymph vessels. Moreover, markers for blood endothelia (monoclonal antibody PAL-E, Factor VIII-related antigen) and markers for cords (strong major histocompatibility complex class II expression on nonrandomly arranged, hairy-appearing cells) were expressed in a mutually exclusive pattern. 3) On epidermal sheets we failed to detect gross changes in the levels of expression of adhesion molecules (CD44, CD54/ ICAM-1, E-cadherin) on keratinocytes in the course of the culture period. The reactivity of a part of the DC in the dermal cords with Birbeck granule-specific monoclonal antibody "Lag" suggests that the migratory population is composed of both epidermal Langerhans cells and dermal DC. We conclude that this organ culture model may prove helpful in resolving pathways and mechanisms of DC migration.

Cell Movement↗
PubMed1996

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

Amino Acid Sequence↗