The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-1... more The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.
This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had ... more This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites. Wir präsentieren eine Fallstudie von Kala-Azar mit gleichzeitiger kutaner Leishmaniose, Die Patientin hatte Fieber, Hepatosplenomegalie und einen Ulcus an der einen Wange. Sie sprach auf Pentostam an. Die Analyse der Isoenzym-Profile der Parasiten aus einer Knochenmarks-Punktion sowie aus dem Ulcus wiesen nach, dass es sich dabei um L. donovani und um L. major handelte. Diese Studie ist die erste Beschreibung einer simultanen Infektion mit L. donovani und mit L. major im Irak. Notre étude rapporte un cas de Kala-Azar accompagné d'une leishmaniose cutanée. La patiente avait la fièvre, une hépatosplénomégalie et un ulcère à une de ses joux. L'infection répondait bien à la Pentostame. L'analyse des isoenzymes montrait que les parasites isolés de la moile osseuse représentaient L. donovani, pendant que ceux de la lésion cutanée représentaient L. major. Notre rapport constitue la première documentation d'un cas d'une infection simultanée par L. donovani et L. major en Iraq.
This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had ... more This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites. Wir präsentieren eine Fallstudie von Kala-Azar mit gleichzeitiger kutaner Leishmaniose, Die Patientin hatte Fieber, Hepatosplenomegalie und einen Ulcus an der einen Wange. Sie sprach auf Pentostam an. Die Analyse der Isoenzym-Profile der Parasiten aus einer Knochenmarks-Punktion sowie aus dem Ulcus wiesen nach, dass es sich dabei um L. donovani und um L. major handelte. Diese Studie ist die erste Beschreibung einer simultanen Infektion mit L. donovani und mit L. major im Irak. Notre étude rapporte un cas de Kala-Azar accompagné d'une leishmaniose cutanée. La patiente avait la fièvre, une hépatosplénomégalie et un ulcère à une de ses joux. L'infection répondait bien à la Pentostame. L'analyse des isoenzymes montrait que les parasites isolés de la moile osseuse représentaient L. donovani, pendant que ceux de la lésion cutanée représentaient L. major. Notre rapport constitue la première documentation d'un cas d'une infection simultanée par L. donovani et L. major en Iraq.
... J. Food Sci.1974, 39, 568−571. [CrossRef]. (13) Bosset, J.-O.; Buetikofer, U.; Fuchs, D.; Imh... more ... J. Food Sci.1974, 39, 568−571. [CrossRef]. (13) Bosset, J.-O.; Buetikofer, U.; Fuchs, D.; Imhof, M.-I.; Tagliaferri, E. Overview and ... Maria DC Antunes, Susana Dandlen, Ana M. Cavaco and Gra a Miguel. Journal of Agricultural and Food Chemistry 2010 58 (10), 6173-6181. ...
Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwif... more Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (KI) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The KI (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESLNSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of ∼11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrus×paradisi (52% identity). This is the first report of phytocystatins from the Ericales.Kiwifruit cystatins were purified from seeds and cortex. Inhibition kinetics and predicted full length peptides from an in-house generated kiwifruit EST database are reported.
In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhi... more In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhibitory activity (PIA) in an abundant globulin fraction extracted in high salt buffer with a Mr of ∼148 kDa by gel-filtration. On a SDS-polyacrylamide gel, a major protein band of 54 kDa was observed under non-reducing conditions. This band was largely replaced by two subunits of Mr 33.5 and 20 kDa under reducing conditions. N-terminal sequencing of the smaller subunit, which had associated PIA, revealed a β-subunit of the 11S globulin-like protein (11S-GLP), legumin. After trypsin, chymotrypsin or papain digestion, the α-subunit of the kiwifruit legumin (11S-GLP) was degraded to varying degrees but there was no effect on the β-subunit, or on the PIA. This 11S-GLP also appeared to inhibit bovine spleen cathepsin D, Candida albicans secreted aspartic proteinases (SAPs) 1, 2 and 4, the plant fungus Glomerella cingulata SAP as well as apple seed aspartic proteinase, but to a much lesser extent kiwifruit seed aspartic proteinase. Through kinetic analysis of pepsin inhibition, the 11S-GLP and the purified β-subunit were found to fit a Michaelis–Menten model for competitive inhibition rather than a tight-binding model characteristic for typical proteinase inhibitors. Extrapolated complete inhibition was only obtained at an 11S-GLP concentration ∼900 times that of the pepsin. Further investigation revealed that the 11S-GLP and the β-subunit acted as weak alternative substrates for pepsin. As the 11S-GLP or the β-subunit were degraded, the PIA activity declined in parallel. We discuss these results in terms of the substrate recognition site of pepsin compared with other proteinases.
Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for... more Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for histological and histopathological characterization. A histochemical study of alkaline phosphatase (ALP) was carried out using the simultaneous azo coupling method, and biochemical studies were performed using disodium phenylphosphate as substrate. Full-term, normal placentae were used for comparison. The specific activity of ALP from cancerous laryngeal tissue was 8.9 mKAU/mg protein compared with 154.7 mKAU/mg protein in the placenta. The ALP was localized histochemically in tumor cells (tumor-specific), blood vessels (vascular) and fibrous tissue (interstitial). The tumor-specific phosphatase was sensitive to inhibition by L-phenylalanine, L-leucine and to a lesser degree by L-tryptophan and levamisole. Placental ALP, on the other hand, was completely inhibited by levamisole, more resistant to leucine and more sensitive to phenylalanine and tryptophan. Biochemical estimation of ALP in cancerous laryngeal tissue combined with inhibition studies revealed that the tumor-specific activity of ALP constitutes 15% of the total ALP activity while the major isoenzyme was the vascular ALP, and around one-third of ALP activity was attributed to the interstitial enzyme. The characterization and localization of these isoenzymes are described and compared with that of the placenta. The significance and implications of the above findings are presented.
Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in pat... more Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.
The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-1... more The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.
This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had ... more This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites. Wir präsentieren eine Fallstudie von Kala-Azar mit gleichzeitiger kutaner Leishmaniose, Die Patientin hatte Fieber, Hepatosplenomegalie und einen Ulcus an der einen Wange. Sie sprach auf Pentostam an. Die Analyse der Isoenzym-Profile der Parasiten aus einer Knochenmarks-Punktion sowie aus dem Ulcus wiesen nach, dass es sich dabei um L. donovani und um L. major handelte. Diese Studie ist die erste Beschreibung einer simultanen Infektion mit L. donovani und mit L. major im Irak. Notre étude rapporte un cas de Kala-Azar accompagné d'une leishmaniose cutanée. La patiente avait la fièvre, une hépatosplénomégalie et un ulcère à une de ses joux. L'infection répondait bien à la Pentostame. L'analyse des isoenzymes montrait que les parasites isolés de la moile osseuse représentaient L. donovani, pendant que ceux de la lésion cutanée représentaient L. major. Notre rapport constitue la première documentation d'un cas d'une infection simultanée par L. donovani et L. major en Iraq.
This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had ... more This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites. Wir präsentieren eine Fallstudie von Kala-Azar mit gleichzeitiger kutaner Leishmaniose, Die Patientin hatte Fieber, Hepatosplenomegalie und einen Ulcus an der einen Wange. Sie sprach auf Pentostam an. Die Analyse der Isoenzym-Profile der Parasiten aus einer Knochenmarks-Punktion sowie aus dem Ulcus wiesen nach, dass es sich dabei um L. donovani und um L. major handelte. Diese Studie ist die erste Beschreibung einer simultanen Infektion mit L. donovani und mit L. major im Irak. Notre étude rapporte un cas de Kala-Azar accompagné d'une leishmaniose cutanée. La patiente avait la fièvre, une hépatosplénomégalie et un ulcère à une de ses joux. L'infection répondait bien à la Pentostame. L'analyse des isoenzymes montrait que les parasites isolés de la moile osseuse représentaient L. donovani, pendant que ceux de la lésion cutanée représentaient L. major. Notre rapport constitue la première documentation d'un cas d'une infection simultanée par L. donovani et L. major en Iraq.
... J. Food Sci.1974, 39, 568−571. [CrossRef]. (13) Bosset, J.-O.; Buetikofer, U.; Fuchs, D.; Imh... more ... J. Food Sci.1974, 39, 568−571. [CrossRef]. (13) Bosset, J.-O.; Buetikofer, U.; Fuchs, D.; Imhof, M.-I.; Tagliaferri, E. Overview and ... Maria DC Antunes, Susana Dandlen, Ana M. Cavaco and Gra a Miguel. Journal of Agricultural and Food Chemistry 2010 58 (10), 6173-6181. ...
Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwif... more Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (KI) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The KI (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESLNSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of ∼11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrus×paradisi (52% identity). This is the first report of phytocystatins from the Ericales.Kiwifruit cystatins were purified from seeds and cortex. Inhibition kinetics and predicted full length peptides from an in-house generated kiwifruit EST database are reported.
In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhi... more In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhibitory activity (PIA) in an abundant globulin fraction extracted in high salt buffer with a Mr of ∼148 kDa by gel-filtration. On a SDS-polyacrylamide gel, a major protein band of 54 kDa was observed under non-reducing conditions. This band was largely replaced by two subunits of Mr 33.5 and 20 kDa under reducing conditions. N-terminal sequencing of the smaller subunit, which had associated PIA, revealed a β-subunit of the 11S globulin-like protein (11S-GLP), legumin. After trypsin, chymotrypsin or papain digestion, the α-subunit of the kiwifruit legumin (11S-GLP) was degraded to varying degrees but there was no effect on the β-subunit, or on the PIA. This 11S-GLP also appeared to inhibit bovine spleen cathepsin D, Candida albicans secreted aspartic proteinases (SAPs) 1, 2 and 4, the plant fungus Glomerella cingulata SAP as well as apple seed aspartic proteinase, but to a much lesser extent kiwifruit seed aspartic proteinase. Through kinetic analysis of pepsin inhibition, the 11S-GLP and the purified β-subunit were found to fit a Michaelis–Menten model for competitive inhibition rather than a tight-binding model characteristic for typical proteinase inhibitors. Extrapolated complete inhibition was only obtained at an 11S-GLP concentration ∼900 times that of the pepsin. Further investigation revealed that the 11S-GLP and the β-subunit acted as weak alternative substrates for pepsin. As the 11S-GLP or the β-subunit were degraded, the PIA activity declined in parallel. We discuss these results in terms of the substrate recognition site of pepsin compared with other proteinases.
Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for... more Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for histological and histopathological characterization. A histochemical study of alkaline phosphatase (ALP) was carried out using the simultaneous azo coupling method, and biochemical studies were performed using disodium phenylphosphate as substrate. Full-term, normal placentae were used for comparison. The specific activity of ALP from cancerous laryngeal tissue was 8.9 mKAU/mg protein compared with 154.7 mKAU/mg protein in the placenta. The ALP was localized histochemically in tumor cells (tumor-specific), blood vessels (vascular) and fibrous tissue (interstitial). The tumor-specific phosphatase was sensitive to inhibition by L-phenylalanine, L-leucine and to a lesser degree by L-tryptophan and levamisole. Placental ALP, on the other hand, was completely inhibited by levamisole, more resistant to leucine and more sensitive to phenylalanine and tryptophan. Biochemical estimation of ALP in cancerous laryngeal tissue combined with inhibition studies revealed that the tumor-specific activity of ALP constitutes 15% of the total ALP activity while the major isoenzyme was the vascular ALP, and around one-third of ALP activity was attributed to the interstitial enzyme. The characterization and localization of these isoenzymes are described and compared with that of the placenta. The significance and implications of the above findings are presented.
Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in pat... more Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.
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