[Medication reconciliation in a department of internal medicine and infectious and tropical diseases: Feedback after one year practice]. / Conciliation médicamenteuse d'entrée en service de médecine interne : retour d'expérience après un an de pratique.

Since April 2015, medication reconciliation is performed in our Department. The objective of this study is to assess the impact of this activity on patients' care after one year of practice. METHODS: All patients who received medication reconciliation between April-October 2015 and June-December 2016 were included in this retrospective study. Undocumented unintentional discrepancies (DNIND) which result from the comparison between the patient's usual treatments and the medication prescribed at admission were collected. Then, a multidisciplinary discussion was initiated to correct them. The gravity of each DNIND was determined a posteriori. RESULTS: A statistical comparison between the two studies (2015 vs. 2016) showed the following significant results: decrease in DNIND (0.9 vs. 0.43), in percentage of patients with at least one DNIND (43% vs 31% P <5.10-6), in reconciliation time (43min vs. 23min) and no significant difference in the distribution of DNIND typology. The main therapeutic classes are: metabolism-diabetes-nutrition (21%), cardiology (18%), pneumology (17%) and neurology-psychiatry (15%). Drugs mainly concerned with DNIND are inhaled anti-asthmatics (13% of the medicines with DNIND), vitamins (8% of DNIND) and the levetiracetam antiepileptic drug (5% of DNIND). CONCLUSION: The implementation of the reconciliation medication allowed a significant reduction of the DNIND that permits to improve the patient healthcare pathway.

Influence of sampling technique and bedding type on the milk microbiota: Results of a pilot study.

The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.

Advanced Modeled Iterative Reconstruction in Low-Tube-Voltage Contrast-Enhanced Neck CT: Evaluation of Objective and Subjective Image Quality.

BACKGROUND AND PURPOSE: Dose-saving techniques in neck CT cause increased image noise that can be counteracted by iterative reconstruction. Our aim was to evaluate the image quality of advanced modeled iterative reconstruction (ADMIRE) in contrast-enhanced low-tube-voltage neck CT. MATERIALS AND METHODS: Sixty-one patients underwent 90-kV(peak) neck CT by using third-generation 192-section dual-source CT. Image series were reconstructed with standard filtered back-projection and ADMIRE strength levels 1, 3, and 5. Attenuation and noise of the sternocleidomastoid muscle, internal jugular vein, submandibular gland, tongue, subscapularis muscle, and cervical fat were measured. Signal-to-noise and contrast-to-noise ratios were calculated. Two radiologists assessed image noise, image contrast, delineation of smaller structures, and overall diagnostic acceptability. Interobserver agreement was calculated. RESULTS: Image noise was significantly reduced by using ADMIRE compared with filtered back-projection with the lowest noise observed in ADMIRE 5 (filtered back-projection, 9.4 ± 2.4 Hounsfield units [HU]; ADMIRE 1, 8.3 ± 2.8 HU; ADMIRE 3, 6.7 ± 2.0 HU; ADMIRE 5, 5.4 ± 1.7 HU; all, P < .001). Sternocleidomastoid SNR and internal jugular vein-sternocleidomastoid contrast-to-noise ratios were significantly higher for ADMIRE with the best results in ADMIRE 5 (all, P < .001). Subjective image quality and image contrast of ADMIRE 3 and 5 were consistently rated better than those for filtered back-projection and ADMIRE 1 (all, P < .001). Image noise was rated highest for ADMIRE 5 (all, P < .005). Delineation of smaller structures was voted higher in all ADMIRE strength levels compared with filtered back-projection (P < .001). Global interobserver agreement was good (0.75). CONCLUSIONS: Contrast-enhanced 90-kVp neck CT is feasible, and ADMIRE 5 shows superior objective image quality compared with filtered back-projection. ADMIRE 3 and 5 show the best subjective image quality.

Adaptation to various sources of dietary sulfur by ruminants.

The objective of these experiments was to determine adaptation by ruminants to dietary sulfur. In Exp. 1, lambs (n = 54; BW = 33.6 ± 0.4 kg) were allotted to 3 treatments: 1) 0% added dietary S (0%S), 2) 0.2% added dietary S (0.2%S), or 3) 0.4% added dietary S (0.4%S). Sulfur was added to the diet as Na(2)SO(4). Lambs fed the 0.2%S and 0.4%S diets had greater (P < 0.01) ADG and G:F compared to those fed the 0%S diet. There was time × diet interaction (P < 0.01) on ruminal hydrogen sulfide gas (H(2)S) concentrations. Ruminal H(2)S was not detected in lambs fed 0%S at any time. Ruminal H2S were not affected (P > 0.19) by diet on d 1 or 8; however, H(2)S were greater (P < 0.01) for lambs fed 0.2%S and 0.4%S than for lambs fed 0%S on d 15, 22, and 29 (0.2% was 931, 846, and 1,131 mg/L and 0.4% was 975, 737, and 1,495 mg/L on d 15, 22, and 29, respectively). These data suggest it takes at least 29 d for peak ruminal H(2)S to occur after exposure to Na(2)SO(4). In Exp. 2, lambs (n = 66; BW = 51.1 ± 0.4 kg) were allotted to 3 treatments: 1) 60% dried distillers grains with solubles (DDGS), 2) corn-based diet with Na(2)SO(4), or 3) corn-based diet with H(2)SO(4). All diets were formulated to contain 0.4%S. Lambs fed Na(2)SO(4) had greater (P < 0.05) ADG, DMI, and G:F than those fed H(2)SO(4) or 60% DDGS. A time × diet interaction occurred (P < 0.01) for ruminal H(2)S. There was no difference (P = 0.82) in H(2)S of lambs on d 1. However, at d 14 and 27 lambs fed supplemental Na(2)SO(4) had the lowest H(2)S concentrations while lambs fed 60% DDGS had the greatest (P < 0.01 on both d); lambs fed H(2)SO(4) were intermediate and different than both. These data suggest that at the same dietary S concentration, acidic S sources increased H(2)S and decreased DMI and ADG. In Exp. 3, Angus cross calves (n = 72; average initial BW = 324 ± 3 kg) were allotted to 3 treatments: 1) corn-based control d 0 through 85 (0%DDGS), 2) gradual step up to 60% DDGS diet (20% DDGS d 0 to 6, 40% DDGS d 7 to 13, 50% DDGS d 14 to 20, and 60% DDGS d 21 to 85; Step-up), or 3) 60% DDGS d 0 to 85 (60%DDGS). Overall, cattle fed 0%DDGS had increased (P < 0.05) DMI and ADG compared with those fed 60%DDGS or Step-up, and G:F was not affected (P = 0.42) by dietary treatment. On d 14, ruminal H(2)S concentrations were greater (P < 0.01) for cattle fed 60%DDGS and Step-up than for those fed 0%DDGS, and they did not differ (P ≥ 0.22) between DDGS-containing diets. These data illustrate that source of S impacts ruminal S metabolism and that S from DDGS is more readily reduced than S from Na(2)SO(4) or H(2)SO(4).

Short communication: antimicrobial susceptibility and frequency of resistance genes in Escherichia coli isolated from bovine mastitis.

Escherichia coli isolated from bovine milk samples submitted to the Ohio Agricultural Research and Development Center Mastitis Laboratory (Wooster) in 1985 to 1987 and in 2009 were compared for antimicrobial susceptibility and prevalence of antimicrobial resistance genes. Forty-four isolates from 1985 to 1987 and 55 isolates from 2009 were tested. Minimum inhibitory concentrations of 15 antimicrobials were determined using a commercially available broth microdilution system. Multiplex polymerase chain reaction was performed for gene detection. The percentage of isolates susceptible to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin was lower in those collected in 1985 to 1987 than in isolates collected in 2009. Susceptibility did not differ between isolates from 1985 to 1987 and isolates from 2009 for the 12 other antimicrobials tested. A trimethoprim/sulfamethoxazole resistance gene was detected more frequently in isolates from 1985 to 1987 than in isolates from 2009. Gene frequencies for streptomycin resistance and tetracycline resistance were similar among 1985 to 1987 isolates and 2009 isolates. Resistance to most antimicrobials did not differ between isolates submitted to a mastitis diagnostic laboratory in 1985 to 1987 and those submitted in 2009. Changes observed indicated an increase in frequency of susceptibility in isolates to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin in 2009 isolates compared with 1985 to 1987 isolates.

Quantitative measurement of genome-wide DNA methylation by a reliable and cost-efficient enzyme-linked immunosorbent assay technique.

DNA methylation, the conversion of cytosine to 5-methylcytosine, is an important epigenetic modification involved in gene regulation. DNA methylation is essential for normal development whereas abnormal methylation has been implicated in pathological conditions including cancer. To evaluate the extent and variation of genome-wide DNA methylation and its changes during cellular differentiation and tumorgenesis as well as the interplay with histone modifications, accurate and reproducible quantification of the genomic DNA methylation level is required. These measurements have so far been achieved only by sophisticated and costly techniques. Here we report the generation of an enzyme-linked immunosorbent assay (methDNA-ELISA) for the accurate quantification of global DNA methylation levels. The linear region of this methDNA-ELISA ranges from 1 to 10%, making it highly suitable for the typical ranges from 2 to 6% in mammalian genomes. This method requires 10 ng of isolated DNA per sample, thus permitting investigation with minimal amounts of DNA previously not applicable for global DNA methylation analysis, e.g., clinical biopsies or cells collected by microdissection.

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations.

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP(C) and the pathological isoform PrP(Sc). The conformational change of the alpha-helical PrP(C) into beta-sheet-rich PrP(Sc) is the fundamental event of prion disease. The transition of recombinant PrP from a PrP(C)-like into a PrP(Sc)-like conformation can be induced in vitro by submicellar concentrations of SDS. An alpha-helical dimer was identified that might represent either the native state of PrP(C) or the first step from the monomeric PrP(C) to highly aggregated PrP(Sc). In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: a novel contributing mechanism of drug-induced-lupus.

UNLABELLED: A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined. OBJECTIVE: To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL. METHODS: Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis. RESULTS: Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen. CONCLUSION: Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.

Static properties of end-tethered polymers in good solution: a comparison between different models.

We present a comparison between results, obtained from different simulation models, for the static properties of end-tethered polymer layers in good solvent. Our analysis includes data from two previous studies--the bond fluctuation model of Wittmer et al. [J. Chem. Phys. 101, 4379 (1994)] and the off-lattice bead-spring model of Grest and Murat [Macromolecules 26, 3108 (1993)]. Additionally, we explore the properties of a similar off-lattice model simulated close to the Theta temperature. We show that the data for the bond fluctuation and the Grest-Murat model can be analyzed in terms of scaling theory because chains are swollen inside the Pincus blob. In the vicinity of the Theta point the structure of the chains is essentially Gaussian in the Pincus blob. Therefore, the data for the second off-lattice model can be compared quantitatively to the self-consistent field theory. Different ways to determine the parameters of the self-consistent field theory are discussed.

Successful palliation of stenosing anorectal melanoma by intratumoral injections with natural interferon-beta.

Anorectal malignant melanoma is an uncommon tumour. Unlike for cutaneous melanoma, there are few guidelines for its optimal management. In particular, very few palliative treatment strategies have been described for patients with advanced disease. We report on an 80 year old patient with locally advanced anorectal melanoma nearly completely blocking the anal orifice and disseminated metastases. Complete regression of the primary tumour and partial remission of the metastases was achieved with intratumoral injections of natural interferon-beta and systemic administration of dacarbazine. The quality of life in this patient was improved markedly by providing relief from severe rectal pain and bleeding. We propose that conservative treatment strategies such as intratumoral injections with interferon-beta should be considered as a palliative treatment option for stenosing anorectal melanoma before an abdominoperineal resection is recommended.

Association of the MIC-A gene and HLA-B51 with Behçet's disease in Arabs and non-Ashkenazi Jews in Israel.

BACKGROUND: Behçet's disease is known to be strongly associated with HLA-B51 in many different ethnic groups. Recently, it was suggested that MIC-A (major histocompatibility complex class I related gene A) is the pathogenic gene after strong association was found between the MIC-A A6 allele of the transmembrane region and the disease in Japanese and Greek patients, although in Greek patients this association was found to be due to linkage disequilibrium with HLA-B51. OBJECTIVES: To investigate microsatellite polymorphism in Arab and non-Ashkenazi Jewish (NAJ) patients in Israel, to determine whether this association occurs in these groups with Behçet's disease, and elucidate the associated HLA allele of the disease. METHODS: Forty four Israeli patients with Behçet's disease, including 20 Arabs and 24 NAJ, and 130 ethnically matched healthy controls were examined for MIC-A microsatellite polymorphism of the transmembrane region using polymerase chain reaction, autoradiography, and sequence analysis. RESULTS: The MIC-A A6 allele was significantly more frequent in the Arab patient group (19/20 (95%)) than in healthy Arab controls (25/42 (60%)) (p(corr)=0.015, OR=12.92), but not in the NAJ patients (16/24 (67%)) compared with NAJ healthy controls (48 /88 (55%)) (p(corr)=1.02, OR=1.667). In stratification analysis of the Arab subgroup, on the confounding effect of MIC-A A6 on HLA-B51 association and vice versa, Behçet's disease was distinctly associated only with HLA-B51. CONCLUSIONS: These results imply strong association between the MIC-A A6 allele and the disease in Israeli Arabs, but not in Israeli NAJ patients. The stratification analysis indicates that this association results secondarily from a strong linkage disequilibrium with HLA-B51, and the real disease susceptibility gene which plays a part in the development of Behçet's disease is most probably the HLA-B51 allele itself.

Neutral loss analysis in MALDI-MS using an ion-gate scanning mode.

Matrix-assisted laser desorption/ionization reflector time-of-flight (MALDI-reTOF) and electrospray ionization (ESI) mass spectrometry (MS) have become essential tools for the characterization of peptides and proteins. Whereas ESI in combination with a triple quadrupole analyzer allows product ion, precursor ion, and neutral loss analyses, MALDI-reTOF instruments can only be used to record product ion spectra based on the in-source or postsource decay (PSD). We describe a new method to perform neutral loss analyses in MALDI-reTOF instruments in a manner that identifies posttranslationally modified peptides and furthermore retrieves sequence information from peptides. The method is based on the selection of ions in a small time interval to record only signals within the corresponding mass interval. By stepping the time interval through the complete mass range, we obtained a spectrum of stable ions by combining the signals of all individually recorded time intervals. This method furthermore permits PSD fragment ions to be identified, since they reach the detector earlier than the stable ions transmitted in the chosen time interval. The neutral loss analysis were calculated by correlating the PSD fragment ions to the corresponding parent ion detected in this time interval. Moreover, this MALDI-MS mode increased the number of detectable signals in complex peptide mixtures and the signal-to-noise ratio.

Reduced body fat and increased hepatic lipid synthesis in mice bearing interleukin-6-secreting tumor.

Chronic secretion of interleukin-6 (IL-6) in mice causes metabolic alteration in the liver, leading to increased synthesis of hepatic cholesterol and fatty acids (FA). Mice were injected with allogeneic tumor cells transduced with the murine IL-6 gene. During the 3 wk after tumor inoculation, elevated serum IL-6 levels were associated with increased spleen and liver weight. Histological examination of sections from the liver showed increased hepatocyte proliferation, resulting in liver enlargement. Body composition analysis revealed that IL-6 caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic de novo synthesis of FA and cholesterol, as measured by (3)H(2)O incorporation, was three to five times as high in mice secreting IL-6 (IL-6 mice) as in pair-fed mice bearing nonsecreting tumors. This increase in FA and cholesterol synthesis is sufficient to maintain hepatic triglyceride secretion at levels comparable with those of pair-fed mice bearing nonsecreting tumors and, presumably, is the main source of cholesterol and FA-phospholipids necessary for hepatocyte proliferation.

Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis.

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.

Molecular properties and involvement of heparanase in cancer progression and normal development.

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.

Aleukemic leukemia cutis presenting as benign-appearing exanthema.

Aleukemic leukemia cutis is a rare condition characterized by the infiltration of the skin by leukemic cells before their appearance in the peripheral blood or bone marrow. We report here a 62-year-old seemingly healthy patient who presented with disseminated erythematous maculae. A skin biopsy showed leukemia cutis of monocytic type. No involvement of bone marrow or peripheral blood was found. The patient developed acute monocytic leukemia 7 months later. We present this case to illustrate how leukemia cutis can masquerade as a clinically benign-appearing cutaneous eruption without leukemic changes in blood or bone marrow. To confirm the diagnosis of aleukemic leukemia cutis, immunohistochemistry of the skin lesions as well as a complete staging procedure is necessary.

Contribution of BDNF-mediated inhibition in patterning avian skin innervation.

Multiple factors are involved in the development and regulation of sensory innervation in skin. The findings we report here suggest that brain-derived neurotrophic factor (BDNF)-mediated inhibition may play an important role in determining the pattern of sensory innervation in avian skin. In birds, cutaneous innervation is restricted to dermis, where axons form a ring of innervation around the base of each feather. Here we show that both BDNF message and protein are more abundant in avian epidermis than dermis when innervation is being established; the BDNF in dermis is localized to feather buds. In vitro, BDNF caused growth cones of NGF-dependent dorsal root ganglion neurons to collapse. Similarly, outgrowth of neurites toward BDNF-secreting fibroblasts was inhibited. The inhibitory effects of BDNF appear to be mediated by the low-affinity p75 neurotrophin receptor, rather than a trk receptor. Thus, the distribution of BDNF in embryonic avian skin and the inhibitory effects of BDNF on cutaneous neurites in vitro suggest that BDNF may be important in restricting axons from entering the epidermis and the core of feather buds during development in vivo.

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.