Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

The effect of temperature and salt concentration on the circular dichroism exhibited by unionized derivatives of L-alanine in aqueous solution

The circular dichroism of Ac–Ala–NHMe, cyclo(–Ala–Ala–), Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe has been measured in water and in aqueous salt solutions as a function of temperature. Only cyclo(–Ala–Ala–) exhibits circular dichroism which is independent of temperature. Each of the linear derivatives of L -alanine exhibits a positive circular dichroism in the range 208–218 nm at 15°C in water. Heating reduces the intensity of the positive circular dichroism, and only Ac–Ala–OMe retains positive circular dichroism at 75°C in water. Isothermal addition of salts produces changes in the circular dichroism of linear derivatives of L -alanine which resemble those seen on heating. The relative effectiveness of the salts tested, at a concentration of 4M, is LiCl ? KCl = NaCl < MgCl₂ ? CaCl₂ ? NaClO₄. The circular dichroism of cyclo(–Ala–Ala–) is also affected by the salts. Extrapolation of the results obtained with Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe to a long polypeptide with a –CH₂R side chain in the L -configuration leads to the conclusion that this polypeptide should exhibit a temperature-dependent salt-sensitive positive circular dichroism between 208 and 218 nm when it exists as a statstical coil.     

Replication and extension of association of choline acetyltransferase with nicotine dependence in European and African American smokers

Choline acetyltransferase is critical in the synthesis of acetylcholine and regulation of cholinergic neuron functions. We recently reported association of the encoding gene ChAT with both smoking cessation and nicotine dependence (ND) in two independent European American (EA) samples; however, in the replication sample, only limited SNPs partially covering the gene were examined. In this study, we examined the association of 14 SNPs, which cover the entire gene, with ND, assessed by smoking quantity (SQ), heaviness of smoking index (HSI), and Fagerström Test for ND (FTND), in 2,037 subjects from 602 families of African American (AA) or EA origin. Individual SNP-based association analysis revealed that five SNPs showed nominal association with at least one ND measure in one of the samples (P = 0.022–0.042); none remained significant after correction for multiple testing. Haplotype-based association analysis revealed that haplotypes G–G–A–C, formed by rs1880676–rs3810950–rs10082479–rs8178990 (P = 0.005–0.0178), and G–G–T–C–G–C, formed by rs1880676–rs3810950–rs10082479–rs8178990–rs3793790–rs12266458 (P = 0.00247–0.00468), displayed significant association with all three ND measures in the AA sample, as did haplotype T–C–G–A–T, formed by rs12266458–rs11101191–rs8178991–rs4838544–rs4838547 (P = 0.00741–0.0103), in the EA sample. All these detected haplotype-based associations remained significant after correction for all major haplotypes for a given SNP combination. Together, our findings, in conjunction with the previous report of the association, warrant further investigation of ChAT in ND.     

The Status of the Hemostasis System under the Influence of Proline Peptides during the Development of Experimental Metabolic Syndrome

A comparative study of the influence of regulatory proline peptides Pro–Gly–Pro, Pro–Arg–Pro, Pro–Gly–Pro–Leu, and Arg–Pro–Gly–Pro on the state of the hemostasis system was carried out in an experiment on male rats with metabolic syndrome. Under these conditions, repeated (7-fold) intranasal administration of the peptides in a daily dose of 50 μg/kg resulted in an increase in the anticoagulant potential of the blood, namely, in an increase in the anticoagulant, fibrinolytic, and antiplatelet activity 20 h and 7 days after the last peptide injection. The arginine–containing peptide Arg–Pro–Gly–Pro had the most pronounced and stable effect on haemostasis under these experimental conditions.     

Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane-attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell-intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane-integrated phosphorylated L1–30. Phase separation experiments with Triton X-114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane-anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1-containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.     

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus)

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1–4)Glc (lactose), Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)Glc (B-tetrasaccharide), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]Glc (B-pentasaccharide), Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (difucosyl lacto-N-neotetraose), Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(α1–3)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1–3)[Fuc(α1–2)]Gal), α-Gal epitope (Gal(α1–3)Gal(β1–4)Glc(NAc)) and Lewis x (Gal(β1–4)[Fuc(α1–3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose) and Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose). Two cheetah milks contained Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1–4)Glc-3’-O-sulfate. Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1–1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

     

Specific cleavage of beta-LPH and ACTH by tonin: release of an opiate-like peptide beta-LPH (61-78).

Tonin, a rat enzyme capable of cleaving angiotensinogen, the tetradecapeptide renin substrate and angiotensin I directly to antiotensin II is also shown to cleave beta-lipotropin into beta-LPH 1–50, 1–51, 51–60, 52–60, 61–78 and 79–91, thereby selectively releasing the opiate-like segment beta-LPH 61–78. Its action on ACTH was similar, releasing ACTH 1–8, 1–7, 3–8, 3–7 and 9–39. In both situations the cleavages are of a selective tryptic-chymotryptic type at specific arginine, phenylalanine residues. Comparison of the tonin cleavage with those of trypsin, trypsin in combination with citraconylation of the lysine residues of beta-LPH is made. The data presented show that tonin does not cleave Met-enkephalin and can be used as an enzyme to study the presence of endorphin-like sequences in polypeptides.     

Bathylagus niger sp. nova (Bathylagidae, Salmoniformes) a new species of Bathylagus from the subpolar waters of the Southern Ocean

Bathylagus niger sp. nova from subantarctic waters of the Southern Ocean is described. This species differs from other representatives of the genus in its combination of the following characters: 23–30 (usually 26–27) rakers on the first gill arch, 20–23 rays in the anal fin, 10–12 rays in the dorsal fin, 40–42 scale pockets in the lateral line, 45–48 (usually 46–47) vertebrae, 4–7 (usually 5–6) pyloric caeca, the greatest body depth is in adults 19–22% SL, and the average head is 21–23% SL.     

Phytoremediation Potential of Weeds in Heavy Metal Contaminated Soils of the Bassa Industrial Zone of Douala,Cameroon

Phytoremediation is a promising option for reclaiming soils contaminated with toxic metals, using plants with high potentials for extraction, stabilization and hyperaccumulation. This study was conducted in Cameroon, at the Bassa Industrial Zone of Douala in 2011, to assess the total content of 19 heavy metals and 5 other elements in soils and phytoremediation potential of 12 weeds. Partial extraction was carried out in soil, plant root and shoot samples. Phytoremediation potential was evaluated in terms of the Biological Concentration Factor, Translocation Factor and Biological Accumulation Coefficient. The detectable content of the heavy metals in soils was Cu:70–179, Pb:8–130, Zn:200–971, Ni:74–296, Co:31–90, Mn:1983–4139, V:165–383, Cr:42–1054, Ba:26–239, Sc:21–56, Al:6.11–9.84, Th:7–22, Sr:30–190, La:52–115, Zr:111–341, Y:10–49, Nb:90–172 in mg kg^?1, and Ti:2.73–4.09 and Fe:12–16.24 in wt%. The contamination index revealed that the soils were slightly to heavily contaminated while the geoaccumulation index showed that the soils ranged from unpolluted to highly polluted. The concentration of heavy metals was ranked as Zn > Ni > Cu > V > Mn > Sc > Co > Pb and Cr in the roots and Mn > Zn > Ni > Cu > Sc > Co > V > Pb > Cr > Fe in the shoots. Dissotis rotundifolia and Kyllinga erecta had phytoextraction potentials for Pb and Paspalum orbiculare for Fe. Eleusine indica and K. erecta had phytostabilisation potential for soils contaminated with Cu and Pb, respectively.     

Screening of Trace Metals in the Plasma of Breast Cancer Patients in Comparison with a Healthy Population

The plasmas of breast cancer patients and healthy donors were analyzed for selected trace metals by a flame atomic absorption spectrophotometric method. In the plasma of breast cancer patients, mean concentrations of macronutrients/essential metals, Na, K, Ca, Mg, Fe, and Zn were 3584, 197.0, 30.80, 6.740, 5.266, and 6.170 ppm, respectively, while the mean metal levels in the plasma of healthy donors were 3908, 151.0, 72.40, 17.70, 6.613, and 2.461 ppm, respectively. Average concentrations of Cd, Cr, Cu, Mn, Ni, Pb, Sb, Sr, and Zn were noted to be significantly higher in the plasma of breast cancer patients compared with healthy donors. Very strong mutual correlations (r > 0.70) in the plasma of breast cancer patients were observed between Cd–Pb, Cr–Li, Li–K, Li–Cd, K–Cr, Li–Pb, Cr–Co, Cu–Ni, Co–K, Cd–K, and K–Pb, whereas, Al–Cr, Ca–Zn, Cd–Sb, Cd–Zn, Ca–Mg, Fe–Zn, and Na–Mn exhibited strong relationships (r > 0.60) in the plasma of healthy donors. The cluster analysis revealed considerably different apportionment of trace metals in the two groups of donors. The average metal concentrations of different age groups of the two donor categories were also evaluated, which showed the build-up of Al, Cd, Co, Cr, Mn, Li, Pb, Sb, and Zn in the plasma of breast cancer patients. The role of some trace metals in carcinogenesis is also discussed. The study indicated appreciably different patterns of metal distribution and correlation in the plasma of breast cancer patients in comparison with the healthy population.     

Nature of T lymphocyte recognition of macrophage-associated antigens. III. Definition of the antigenic regions of human fibrinopeptide B involved in guinea pig T-cell responses

The clonal diversity of guinea pig T-lymphocyte responses to the 14-amino-acid peptide antigen human fibrinopeptide B (hFPB, Bβ1–14) and sequential hFPB³ homologs (Bβ5–14 and 7–14) was examined using bromodeoxyuridine (BUdR) and light elimination of T cell responses. PPD and hFPB-immune strain 2 guinea pig T cells and macrophages were stimulated in a first culture with PPD, Bβ1–14, 5–14, or 7–14; BUdR was added on the second day and the cultures exposed to light on the third day. The BUdR and light-treated T cells recovered from the first culture were restimulated in a second culture containing fresh stimulator macrophages and PPD, Bβ1–14, 5–14, and 7–14. BUdR and light-treated T cells initially stimulated with Bβ1–14 in the first culture showed no responsiveness to Bβ1–14, 5–14, or 7–14 in the second culture. BUdR and light-treatment of T cells initially stimulated with Bβ5–14 eliminated 70 to 80% of the subsequent response to Bβ1–14 and all of the responsiveness to Bβ7–14. Similar treatment of T cells stimulated with Bβ7–14 reduced responsiveness to Bβ1–14 by 50 to 60% and to Bβ5–14 by 60 to 70%. These observations indicate that T-cell responses are directed against three antigenic regions in the hFPB molecule; the major region defined by the carboxy-terminal sequence including residues 7 to 14, a second minor antigenic region including residues 5 and 6, and a third minor region including the amino terminal residues 1 to 4. Results are discussed with respect to the regions of the hFPB molecules that are recognized by antigen-binding T-cell receptors and the regions which interact with stimulator macrophages.     

Assessment of Toxic Metal Contamination of Bottom Sediments in Water Bodies in Urban Areas

A study was carried out on 20 water bodies of the same origin in southern Poland. The study objectives included the assessment of toxic metal contamination in the bottom sediments of the water bodies in comparison with the geochemical background and sediments found in the substrate (i.e., vicinity) of the water bodies (i.e., the formations present in the surroundings of the water body itself), thus demonstrating the scale of anthropogenic enrichment of bottom sediments with toxic metals and assessing the cumulative impact on water bodies. The following amounts of toxic metals were found in the bottom sediments of the water bodies examined: 181.7–35200.0 ppm for zinc, 33.3–1648.8 ppm for lead, 1.8–359 ppm for cadmium, 14.0–271.5 ppm for copper, 45.3–167.5 ppm for chromium, and 12–128.5 ppm for nickel. Ratios of the values measured to the geochemical background were as follows: 0.7–135.9 (Zn), 0.6–53.0 (Pb), 0.7–143.6 (Cd), 0.9–18.1 (Cu), 5.0–18.6 (Cr), 1.1–11.7 (Ni).     

Analysis of the interactions of sulfur‐containing amino acids in membrane proteins

The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic or/and aliphatic–aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small‐molecule model systems at the ab‐initio level, predicts that Met–Met interactions are stronger than Met–Cys ≈ Met–Phe ≈ Cys–Phe interactions, stronger than Phe–Phe ≈ Phe–Leu interactions, stronger than the Met–Leu interaction, and stronger than Leu–Leu ≈ Cys–Leu interactions. These results show that sulfur‐containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity.     

Chemical composition of the essential oils of ripe berries of Juniperus oxycedrus L., growing wild in Kosovo

The main objective of this work was to study the essential oil composition of ripe Juniperus oxycedrus L. berries and its natural variation among wild populations in Kosovo. Essential oil was analysed using GC-FID and GC–MS. Plant materials were collected from five locations in Kosovo in August and September of 2011. In total, twenty-seven compounds were identified in the essential oils. The main components were β-myrcene (45.5–56.9%), α-pinene (10.2–36.6%), dl-limonene (3.6–13.8%) and germacrene D (1.7–8.7%). Of the total identified compounds, monoterpenes constituted the highest percentage of all components (70.24–88.22%), followed by oxygenated sesquiterpenes (4.9–11.4%), sesquiterpenes (3.5–11.0%), oxygenated monoterpenes (0.2–2.7%) and oxygenated diterpenes (0.0–1.7). Hierarchical Cluster Analysis (HCA) and Principal Component Analyses (PCA) were used to identify any geographical variations in essential oil composition. Statistical analysis suggests that the clustering of populations is not related to their geographic location, but rather seemed to be linked to local selective forces acting on chemotype diversity.     

Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry

The elemental composition of 10 methanogenic species was determined by inductively coupled plasma emission spectrometry and by a C-H-N-analyzer. The 10 species were representative of all three orders of the methanogens and were cultivated under defined conditions. Special emphasis was given toMethanosarcina barkeri, represented by 5 strains and cultivated on various substrates. The following elements with the lowest and highest values in parentheses were determined: C (37–44%, w/w), H (5.5–6.5%), N (9.5–12,8%); Na (0.3–4.0%), K (0.13–5.0%), S (0.56–1.2%), P (0.5–2.8%), Ca (order I: 85–550 ppm; order II: 1000–4500 ppm), Mg (0.09–0.53%), Fe (0.07–0.28%), Ni (65–180 ppm), Co (10–120 ppm). Mo (10–70 ppm), Zn (50–630 ppm), Cu (<10–160 ppm), Mn (<5–25 ppm). The biggest variations were found with respect to N and K, which both seem to have important physiological functions. Although it is unknown whether zinc and copper are essential trace elements for methanogens, all investigated species contained remarkably high zinc contents, whereas copper seemed to be present only in some species.     

Energetics of protein–DNA interactions: An exact calculation for binding of ligands to a lattice of overlapping sites

Exact equal ions are developed for analyzing the binding of ligands to a linear lattice of overlapping sites in which occupied–unoccupied as well as occupied–occupied interactions are included for the analysis of the binding isotherms. We demonstrate that positive cooperativity on the binding of ligands to multiple sites may derive from either occupied–unoccupied or occupied–occupied interactions. When the binding of proteins to linear polynucleotides and DNA has exhibited positive cooperativity protein–protein (occupied–occupied), interactions have heretofore been invoked as the sole energetic source in determining the cooperative effect. Models and equations developed previously for the analysis of these binding isotherms have included only the protein–protein interactions (usually characterized with the symbol ω). The exact equations of this paper are capable of analyzing binding data in a manner to evaluate the relative importance of both occupied–unoccupied and occupied–occupied interactions Relations derived here are employed to analyze some existing data, and the resulting parameter values are compared to those developed with equations employing only the protein–protein (occupied–occupied) interactions. The resulting parameter values are qualitatively different. Values of the binding constants differ by about three orders of magnitude. When only protein–protein interactions are taken into account, the resulting free energy of interaction is negative, indicating attractive forces between bound protein molecules; when both occupied–unoccupied and occupied–occupied interactions are applied, the resulting free energies of interaction are positive, indicating destabilizing forces acting primarily on the polynucleotide lattice. © 1995 John Wiley & Sons, Inc.     

The efficacy of Dynorphin fragments at the κ, μ and δ opioid receptor in transfected HEK cells and in an animal model of unilateral peripheral inflammation

BackgroundDynorphin 1–17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1–17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, μ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1–17 and fragments (1–6, 1–7 and 1–9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1–7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund’s Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1–7, 1–17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1–7 and 1–9 displayed the greatest activity across all receptor subtypes, while dynorphin 1–7, 1–9 and 1–17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1–7 and U50488H, but not dynorphin 1–17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1–7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1–7 mediates analgesia.     

The effect of click rate on latency and interpeak interval of the brain-stem auditory evoked potentials in children from birth to 6 years

Latency and interpeak interval of the brain-stem auditory evoked potentials at different click rates were measured in 80 healthy children from birth to 6 years, and 21 adults. Clicks were presented at 10, 30, 50, 70 and 90/sec, and 70, 40 and 20 db HL. At high stimulus intensity (70 dB SL), all latencies of waves I, III and V and the I–V, I–III and III–V intervals showed a progressive prolongation with increasing repetition rate. The latency- and the interval-rate functions were similar for all age groups but their slopes were slightly steeper in younger than in older. As click rate increased from 10/sec to 90/sec, the latencies of waves I, III and V at different age groups were prolonged by 4–10%, 9–13% and 12–15% respectively, and the intervals of I–V, I–III and III–V were prolonged by 15–16%, 8–16% and 14–24% respectively. The mean increments of wave V latency and I–V interval in different age groups were 0.404–0.575 and 0.332–0.526 msec respectively with increasing click rate from 10 to 50/sec, and 0.697–1.009 and 0.629–0.776 msec respectively with increasing click rate from 10 to 90/sec. The younger the age the larger the absolute increments for all these BAEP parameters, but the increasing rates for a BAEP measure were similar among different age groups, exhibiting no age-dependent differences. The III–V/I–III interval ration in most age groups was increased by 3–10% with increasing click rate from 10 to 90/sec, suggesting that the III–V interval was affected by stimulus rate slightly more than I–III interval.At moderate (40 dB HL) and low (20 dB SL) intensity, all waves and intervals showed similar latency- and interval-rate functions to those at high intensity. This demonstrates that the shifting latencies and interpeak intervals with increasing click rate appeared to be independent of the stimulus intensities.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.