Effect of Thyrotropin-Releasing Hormone and Its Synthetic Analogue Digipramine on Certain Indices of Hemostasis <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

The effect of neuropeptide thyrotropin-releasing hormone (TRH) and its synthetic analogue digipramine on certain indices of blood coagulation and fibrinolysis were studied in vitro and in vivo. The peptides added to the pool of normal rat plasma at 10⁻¹⁰ to 10⁻³ M increased the procoagulant activity but had virtually no effect on fibrinolysis. Intravenous administration of TRH and digipramine increased the procoagulant activity of the blood and platelet aggregation but decreased fibrinolysis; digipramine had a more pronounced effect on the coagulation potential of the blood and a less pronounced effect on fibrinolytic indices as compared to TRH. Intranasal administration of the peptides did not change the pattern of their effect on indices of hemostasis although the effects became less pronounced.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 311–315.Original Russian Text Copyright © 2005 by Grigorjeva, Golubeva.     

Peptidasen II. Zur Lokalisation der Dipeptidylpeptidase IV (DPP IV). Histochemische und biochemische Untersuchung

Zusammenfassung Histochemisch wird die Dipeptidylpeptidase IV (DPP IV) mit Glycyl-prolyl(Gly-pro)-naphthylamiden als Substraten, stabilen und instabilen Diazoniumsalzen zur Simultankupplung und unterschiedlichen Puffern bei Ratten, Mäusen, Katzen, Meerschweinchen, Kaninchen, Hamstern und in menschlichen Dünndarmbiopsien nach verschiedenen Gewebevorbehandlungen untersucht. Die besten Resultate liefert 1,7–3,4 mM Gly-pro-4-methoxy-2-naphthylamid und 1 mg Fast Blue B/ml und mit Einschränkungen 0,025 ml hexazotiertes Neufuchsin/ml in 0,1 M Cacodylat- oder Phosphat-Puffer, pH 7,5, und frische Kryostatschnitte zum Nachweis der Gesamtaktivität der DPP IV und gefriergetrocknete Schnitte nach Celloidinmontage zur ortsgetreuen Lokalization des Enzyms. Schnitte von aldehydfixiertem Material eignen sich zur Untersuchung des Umsatzes von Gly-pro-naphthylamiden zwischen pH 5 und 7 mit hexazotiertem Neufuchsin oder p-Rosanilin in Lysosomen.Die DPP IV ist fest strukturgebunden und weist Spezies- und Organdifferenzen auf. Im allgemeinen kommt das Enzym in Kapillarendothelien, Sinusoidalzellen, Perineurium, Schalt- und Sekretrohrepithelien, Mikrovillizone von Darmkrypten und-zotten, Uterus, Tube, proximalen Nierentubuli sowie Nebenhodengang, Hepatocyten- und Lymphocytenmembran, Plasmalemm mehrschichtiger und Übergangsepithelien sowie in der Kapsel und im Interstitium zahlreicher Organe vor.Die biochemische Untersuchung der DPP IV wird mit 10 mM Gly-pro-2-naphthylamid in 0.1 M Cacodylat-Puffer, pH 7 durchgeführt und die Enzymaktivität fluorometrisch in Ratten- und Meerschweinchenorganen bestimmt. Die Befunde bestätigen und erweitern die auffälligen spezies- und organabhängigen Unterschiede des histochemischen DPP IV-Nachweises.Verglichen mit anderen Di- sowie Tripeptidyl- und Aminosäurenaphthylamiden deuten die Befunde darauf hin, daß es sich bei der DPP IV um eine Peptidylpeptidase handelt, die neben dem Kollagenabbau an anderen Stoffwechselvorgängen beteiligt ist.

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Histochemical and biochemical distribution of dipeptidylpeptidase IV (DPP IV)

Summary Fresh frozen, unfixed, chloroforme-acetone treated or freeze-dried cryostat sections or sections from aldehyde-fixed blocks of tissue were tried for the histochemical investigation of dipeptidylpeptidase IV (DPP IV) with l-glycyl-l-prolyl(gly-pro)-naphthylamides as substrates and stable or unstable diazonium salts for simultaneous coupling and various buffers, pH 5–7.5 in rats, mice, guinea-pigs, cats, rabbits, hamsters and human enterobiopsies. The best results are obtained with 1.7–3.4 mM gly-pro-4-methoxy-2-naphthylamide and 1 mg Fast Blue B/ml or (with some limitations) 0.025 ml hexazotized new fuchsine/ml in 0.1 M cacodylate or phosphate buffer, pH 7.5 and unfixed sections for the demonstration of the total activity of DPP IV and freeze-dried celloidin-mounted cryostat sections for the precise localization of the enzyme or the detection of lysosomes, Golgi apparatus and secretion granules; sections from aldehyde fixed tissue blocks are only suitable to study the lysosomal hydrolysis of gly-pro-naphthylamides between pH 5 and 7 when hexazotized p-rosaniline or new fuchsine are employed.DPP IV is firmly bound to structures and shows species- and organ-dependent differences. In general, the enzyme occurs in the capillary endothelium, sinusoidal cells, perineurium, epithelial cells of intercalated and striated ducts, microvillous zone of intestinal crypts and villi, uterus, Fallopian tube, ductus epididymis and proximal renal tubules, hepatocyte and lymphocyte membrane, plasmalemma of pseudostratified and transient epithelia and in the capsules and interstitium of many organs. These sites of activity can be completely inhibited by diisopropyl fluorophosphate and partially by Pb²⁺; Mg²⁺, Mn²⁺, Co²⁺ EDTA are without any influence. Phenantrolin may activate DPP IV.The biochemical assay works with 10 mM gly-pro-2-naphthylamide in 0.1 M cacodylate buffer, pH 7; the enzyme activity is determined fluorometrically in guinea-pig and rat organs; the data confirm and enlarge the species-and organ-dependent differences revealed by histochemistry.Compared with other dipeptide as well as tripeptide and amino acid naphthylamides the results obtained for DPP IV suggest a peptidylpeptidase which seems to be involved in other metabolic processes beside the degradation of collagen.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (SFB 105)     

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC–MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C₁₈ analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25–1000 ng/ml (r²≥0.996) with accuracy ≤9.8% and precision ≤13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1300 ng/ml for MPA. This HPLC–MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.     

Intercentral Relations of the Human EEG during Listening to Music

Different musical styles and intensities produce characteristic patterns of changes in the correlation of neocortical biopotentials. Classical music of three intensities and rock music of medium and high intensities were shown to produce asymmetry in the pattern of coherence (Coh) of the cortical activity of listeners: intrahemispheric Coh in the right hemisphere increased, while a decrease in the EEG synchronization prevailed in the left hemisphere. A focus of Coh integration, most pronounced for the γ frequencies, was formed during listening to both classical and rock music (except rock music of a low intensity). Listening to rock music of a low intensity substantially increased the probability of a decrease in intra- and interhemispheric Coh in both hemispheres, which was pronounced in all frequency bands. Listening to rock music changed interhemispheric Coh in a greater percent of cases than listening to classical music.__________Translated from Fiziologiya Cheloveka, Vol. 31, No. 4, 2005, pp. 27–32.Original Russian Text Copyright © 2005 by Sakharov, Davydov, Pavlygina.     

Expression of copper-responsive genes in HepG2 cells

In recent years, considerable emphasis has been focused on identifying new chemopreventive agents, which could be useful for the human population. Piperine is a pure, pungent alkaloid constituent of black and long peppers (piper nigrum and piper longum), which is a most common spice used throughout the world. In the present study, we examined the protective role of piperine during experimental lung carcinogenesis with reference to its effect on DNA damage and detoxification enzyme system. The activities of detoxifying enzymes such as glutathione transferase (GST), quinone reductase (QR) and UDP-glucuronosyl transferase (UDP-GT) were found to be decreased while the hydrogen peroxide level was increased in the lung cancer bearing animals. Supplementation of piperine (50 mg/kg bwt) enhanced the detoxification enzymes and reduced DNA damage as determined by single cell electrophoresis. Furthermore, the DNA-Protein cross links which was found to be high in lung cancer bearing animals was also modulated upon supplementation with piperine. Our present results explain the understanding of unique association between anti-peroxidative effect of piperine and ultimately the capability of piperine to prevent cancer. (Mol Cell Biochem 268: 141–147, 2005)     

Distribution,species diversity and life-form spectra of plant communities along an altitudinal gradient in the northern slopes of Qilianshan Mountains,Gansu, China

We studied the distribution pattern, species diversity and life-formspectra of plant communities along an altitudinal gradient in the mid-sectionofthe northern slopes of Qilianshan Mountains by means of multivariate analyses.Two data sets (167 species × 75 plots, 10 environmental variables ×75 plots), originated from the fieldworks in 1998–1999, were subjected toTWINSPAN and DCCA, resulting in 8 major plant communities: 1)Asterothamnus centraliasiaticus–Halogetonarachnoideus desert grassland on azonal substrates from 1450 to 1600m and 2) zonal Reaumuria soogorica desertgrassland on gravels from 1470 to 1900 m; 3) Stipaprzewalskii–Stipa purpurea montane grassland from 2200 to 2900m; 4) Polygonum viviparum alpine grasslandfrom 2900 to 3700 m; 5) Caraganastenophylla–Ajaniafruticulosa dry-warm shrubland from 2350 to 2800 m; 6)Sabina przewalskii mid-wet warm forest from 2700 to 3300m; 7) Picea crassifolia cold coniferousforestfrom 2450 to 3200 m; 8) Caragana jubatawet-cold alpine shrubland from 3100 to 3700 m. Species diversityand species richness of both grasslands and forests peaked at the intermediateportion of the elevational gradient. Evenness might be strongly influenced byeither biotic or abiotic factors at a local scale, while seems quiteindependentof an elevational gradient at landscape scales. Beta-diversity decreased from1500 to 3700 m, indicating that species turnover declined withincreased elevation. Both richness of life-form and total species richness in agiven altitudinal belt (gamma-diversity) peaked at intermediate elevations,while relative species richness of different life-form varied differently alongthe altitudinal gradient.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure–activity-relationship data obtained in this way led to the preparation of a series of α-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.     

Topography of digestive enzymes in small intestine and activity of corresponding hydrolases in liver and kidney of adult pigs

Distribution of activities of disaccharidases (saccharase, maltase), amino- and dipeptidases, dipeptidyl peptidase IV (DPP) and alkaline phosphatase along the small intestine and of these hydrolases in liver and kidney, as well as distribution of the digestive enzyme activities between the membrane and cytosol fractions of enterocytes is characterized in adult 6-month old pigs. It has been shown that maximum of the studied enzyme activities, except for DPP, takes place in ileum. A higher level of the activities of the soluble form of saccharase, maltase, and aminopeptidase M and a less significant level of the activity of the glycyl-L-leucine dipeptidase soluble form in jejunum and ileum is found than those in rats and monkeys. The obtained data demonstrate the existence of the complex enzyme system in small intestine, liver, and kidney that participate in realization of nutritive and trophic-barrier functions.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 39–43.Original Russian Text Copyright © 2005 by Smirnova, Gordova, Timofeeva, Shcherbakov.     

Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride

An improved HPLC method using a silica gel column with fluorescence detection (excitation at 300 nm and emission at 365 nm) was developed for the determination of sulpiride concentrations in plasma. Analysis of sulpiride in plasma samples was simplified by a one-step liquid–liquid extraction after alkaline treatment of only 1 ml of plasma. The low limit of quantitation was 20 ng/ml with a coefficient of variation of less than 20%. A linear range was found from 20 to 1500 ng/ml. This HPLC method was validated with the precision for inter-day and intra-day runs being 0.36–8.01% and 0.29–5.25%, respectively, and the accuracy (standard deviation of mean, SD) for inter-day and intra-day runs being −1.58 to 5.02% and −2.14 to 5.21%, respectively. Bioequivalence of the two products was evaluated in 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment cross-over study. Sulpiride plasma concentrations were analyzed with this validated HPLC method. Results demonstrated that the two tablet formulations of sulpiride appear to be bioequivalent.     

Forest soil respiration across three climatically distinct chronosequences in Oregon

To assess the relative influence of edaphoclimatic gradients and stand replacing disturbance on the soil respiration of Oregon forests, we measured annual soil respiration at 36 independent forest plots arranged as three replicates of four age classes in each of three climatically distinct forest types. Annual soil respiration for the year 2001 was computed by combining periodic chamber measurements with continuous soil temperature measurements, which were used along with site-specific temperature response curves to interpolate daily soil respiration between dates of direct measurement. Results indicate significant forest type, age, and type × age interaction effects on annual soil respiration. Average annual soil respiration was 1100–1600, 1500–2100, and 500–900 g C m⁻² yr⁻¹ for mesic spruce, montane Douglas-fir, and semi-arid pine forests respectively. Age related trends in annual soil respiration varied between forest types. The variation in annual soil respiration attributable to the climatic differences between forest types was 48%(CV). Once weighted by the age class distribution for each forest type, the variation in annual soil respiration attributable to stand replacing disturbance was 15%(CV). Sensitivity analysis suggests that the regional variation in annual soil respiration is most dependent on summer base rates (i.e. soil respiration normalized to a common temperature) and much less dependent on the site-specific temperature response curves (to which annual rates are relatively insensitive) and soil degree-days (which vary only 10% among plots).     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Inorganic-ion resistance by bacteria isolated from a Mexico City freeway

Bacteria were isolated from soil samples, containing high exchangeable lead concentrations, obtained from a busy freeway in the México City metropolitan area. Forty-five selected strains (86.7% Gram-positive) had a single MIC distribution pattern for lead (800–1600 µg/ml lead nitrate) and were considered lead-resistant. The isolates showed variable levels of resistance to arsenate (86.7%), chromate (66.7%), cadmium (57.6%), and mercury (31.1%) ions. Multiple inorganic-ion resistance was shown by all strains.     

The effect of ampicillin on the electrophysical properties of Escherichia coli cells

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OSs) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10–1000 kHz of the orienting electric field and does not affect the OSs of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 µg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OSs of cells can be used to evaluate their resistance to ampicillin.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 126–131.Original Russian Text Copyright © 2005 by Guliy, Markina, Ignatov, Shchegolev, Zaitseva, Bunin, Ignatov.     

The impact of nitrogen fertilization of tomato on feeding site selection and oviposition by Trialeurodes vaporariorum

The sexual calling incidence of wild and mass-reared Mediterranean fruit fly males, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), was studied at 25 °C and a photophase of 14 h starting at 0600 h. Wild males fed on yeast hydrolysate and sugar attained sexual maturity earlier and exhibited higher calling activity than males fed on sugar alone. In both sugar and yeast hydrolysate-fed wild males, calling levels were high during the first hours of the photophase, declined at 0900–1000 h, then increased again reaching high levels between 1200 and 1500 h, and finally declined and ceased after 1600 h. Food deprivation for 8 h or more caused a significantly lower frequency of calling. Irradiated (sterile) and unirradiated mass-reared males fed on either sugar and yeast or sugar alone did not differ in the age of attaining sexual maturity (maximum levels of calling occurred about 8 days earlier than in the wild males). However, irradiated males fed on sugar called less frequently than males in the other treatments of mass-reared males. The daily rhythm of calling in protein and sugar-fed irradiated males differed somewhat from that of wild males. Calling was recorded at high levels from the beginning of the photophase until 1300 h without any decrease at 0900–1000 h. It then decreased to very low levels from 1500–1800 h and increased slightly at 1900 h. These findings are discussed in relation to the mating behavior of the fly and to the effectiveness of released sterile males in Sterile Insect Technique programs.     

The stimulation of chloride transport by prostaglandins and their interaction with epinephrine,theophylline, and cyclic AMP in the corneal epithelium

Summary Prostaglandins (E₁, E₂ and F₂) stimulated the chloride transport of the frog corneal epithelium with maximal effects at 10^–5 m in the aqueus side. This stimulation does not occur in Cl-free solutions and the net³⁶Cl flux increased proportionally to the short-circuit current. Polyphloretin phosphate (PPP) and diphloretin phosphate (DPP) inhibited the response if added within 3 min before PGE₁. The maximal response to epinephrine 10^–5 m and dibutyryl cyclic AMP 10^–3 m was not changed by further addition of prostaglandins, but these drugs produced their full effect when administered at the peak of the response of prostaglandins. The maximal response to theophylline 10^–5 m was increased by PGE₁. PPP and DPP did not modify the response to epinephrine. Prostaglandin stimulation of the chloride transport was accompanied by increased light transmission through partially opaque corneas. The known release of prostaglandins in the aqueous humor can be associated to a direct action on the corneal epithelium manifested in the activation described herein.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Secondary release of thromboxane A2 in aerosol leukotriene C4-induced bronchoconstriction in guinea pigs

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01–1.0μg/ml of leukotriene C4 and 12.5–400μg/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0μg/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6, 7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole     

Response of microcosm zooplankton to acidification

Zooplankton community transformation in response to soft water acidification was studied in two experimental series using the microcosm method. The dynamics of total abundance, changes in the proportions of the main zooplankton groups, stability of the dominant complex, and species diversity were evaluated. Zooplankton population proved to decrease at all studied low pH (5–6, 4–5, and 3–4) within the first two weeks relative to the control. At water pH 5–6, either Rotifera-Copepoda or Cladocera-Copepoda zooplankton complex was established depending on the initial community composition; while at pH 4–5, Copepoda predominated in the established zooplankton community. The community edificator species was replaced at pH 4–5. The lowest diversity index was also observed at this pH (1.0–1.5 bit/ind.). In addition, the proportion and absolute population of copepod nauplii and cladoceran Scapholeberis mucronata increased at low pH. A decrease in water pH to 3–4 proved to be disruptive for the zooplankton community.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 100–108.Original Russian Text Copyright © 2005 by Kurbatova.     

Are Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA suitable substrates for the demonstration of cathepsin D activity?

Summary The suitability of Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA for the assessment of cathepsin D activity was tested in biochemical and histochemical experiments. Substrates were dissolved in dimethylformamide and used at 0.1–0.5 mM in various buffers over a pH range of 3.5–7.4. Homogenates of various rat organs and isolated purified enzymes [cathepsin D from bovine spleen, dipeptidyl peptidase (DPP) I.V from porcine kidney and rat lung] were used as enzyme sources. Pepstatin, di-isopropylfluorophosphate (DFP),p-chloromercuribenzoate,o-phenanthroline and a series of DPP IV inhibitors were used in inhibitor experiments. At pH 3.5 and 5.0, substrates were used in a two-step postcoupling procedure with aminopeptidase M and dipeptidyl peptidase IV as auxiliary enzymes and Fast Blue BB as coupling agent. Results were compared with those obtained with haemoglobin. Above pH 5.0 substrates were used in a one-step postcoupling procedure.Cryostat sections of snap-frozen or cold aldehyde-fixed tissue pieces of various rat organs and biopsies of human jejunal mucosa were used in histochemical experiments. As in biochemical tests a two-step procedure was used in the pH range 3.5–5.0, but Fast Blue B was used in the second step for the simultancous coupling. Above pH 5.0 a onestep simultaneous azo coupling procedure was used with Fast Blue B as coupling agent.At pH 3.5 the hydrolysis rate of both synthetic substrates was about 100 x lower than that of haemoglobin when cathepsin D from bovine spleen was used. The activity was inhibited by pepstatin. With increasing pH the hydrolysis rate of Z-Arg-Gly-Phe-Phe-Pro-MNA increased, while that of Z-Arg-Gly-Phe-Phe-Leu-MNA decreased when organ homogenates were used as enzyme sources. However, the activity was not inhibited by pepstatin. It was inhibited by DFP. The extent of the inhibition with other substances was species and organ dependent. Z-Arg-Gly-Phe-Phe-Pro-MNA was also cleaved by isolated and purified DPP IV of porcine kidney and rat lung and the activity was inhibited by DFP and DPP IV inhibitors.In histochemical experiments the staining obtained with both synthetic substrates at pH 3.5 was weak and rather diffuse, with only slight accentuation or none at all in the lysosomal region of cells. In the pH range 5.5–7.4 a very distinct reaction was observed with Z-Arg-Gly-Phe-Phe-Pro-MNA only. The reaction product was localized in the brush border of enterocytes and of cells of the proximal kidney tubules. Endothelial cells of glomeruli and capillaries of the propria of the human jejunum also displayed a positive reaction. Lymphocytes in the propria of rat small intestine reacted to some extent. The reaction was inhibited by DFP. The extent of the inhibition with other substances varied.Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA are not efficient substrates for the assessment of cathepsin D activity. In histochemical studies diffusion artifacts must always be considered. In the pH range 5.5–7.4, Z-Arg-Gly-Phe-Phe-Pro-MNA is cleaved by a serine endopeptidase and by a metalloendopeptidase. It remains to be established whether prolyl endopeptidase or DPP IV (or both) and which metalloendopeptidase are responsible for the cleavage. In the evaluation of enterobiopsies the demonstration of this activity is a sensitive means for the assessment of the state of the brush border.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel–nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS–PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH₂-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues −180 to −175, NH₂-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH₂-terminal sequence, LDXNQLY, corresponding to residues −73 to −67 of the proregion peptide and thus were generated by removal of 126 residues from the NH₂-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37°C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k_cat/K_m) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM⁻1 s⁻¹, respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing 2 × 10⁹ High Five cells.