Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

Summary The kinetics of MT reorientation in primary roots ofZea mays cv. Merit, were examined 15,30,45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4–5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10^–9 to 10^–6M IAA. IAA treatment at 10^–8M or less showed no major or consistent changes but 10^–7 M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10^–5M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether) N,N,NN-tetraacetic acid - MTs cortical microtubules - QC quiescent center - MES/TRIS 2-(N-morpholino)ethanesulfonic acid/tris(hydroxymethyl)aminomethane - IAA indole-3-acetic acid - PBS phosphate buffered saline - PHEMD [60 mM Pipes (piperazine-diethanesulfonic acid), 25 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 10 mM EGTA, 2mM MgCl₂ pH7.0 adjusted with NaOH] containing 5% dimethyl sulfoxide     

Plant regeneration from cotyledons of Prunus persica,Prunus domestica,and Prunus cerasus

Shoots were regenerated from the proximal region of immature cotyledons (with the embryonic axis removed) of Prunus persica (peach) and from the same area in mature cotyledons of P. domestica (plum) and P. cerasus (sour cherry) on MS medium containing (in mgl^-1) thiamine-HCl, 0.4; nicotinic acid, 0.5; pyridoxine-HCl, 0.5; sucrose, 25 000; and 0.7% agar. The medium was supplemented with 0.0–2.5 M indole-butyric acid and 5–12.5 M thidiazuron. Cultures were incubated at 24 °C under 16h photoperiod. Shoots regenerated adventitiously over a broad range of thidiazuron concentrations and 2.5 M indole-butyric acid in 35 days. The presence of the embryonic axis inhibited the development of shoots. Regenerated shoots of peach and plum were rooted on half-strength MS inorganic semi-solid medium with 2.5–5.0 M indole-butyric acid. Rooted plants were acclimatized and transferred to the greenhouse.Abbreviations BAP 6-benzylaminopurine - IBA indole-butyric acid - TDZ N-phenyl-N-1,2,3-thidiazol-5-ylurea - IAA indole-acetic acid - NAA -naphthalene-acetic acid     

In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation

An efficient protocol has been developed for the in vitro propagation of Bambusa tulda through shoot proliferation. Shoots from 3-week-old aseptically grown seedlings were used to initiate cultures. Multiple shoots were obtained on liquid Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (8×10^–6M) and kinetin (4×10^–6M). Continuous shoot proliferation at a rate of 4–5 fold every three weeks was achieved through forced axillary branching. More than 90% of the shoots could be rooted on a modified MS medium containing indoleacetic acid (1×10^–5M) and coumarin (6.8×10^–5M). Following simple hardening procedures, the in vitro raised plants were transferred to the soil with more than 80% success.Abbreviations BAP 6-benzylaminopurine - 2-ip 6-,-dimethylallylaminopurine - Kn kinetin - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - NAA 1-naphthaleneacetic acid     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Biotransformations of aromatic diniitriles using Rhodococcus equi cells

Short incubation (1 – 5 h) of aromatic dinitriles with resting cells of Rhodococcus equi A4 produces the corresponding cyano amides in 48 – 81% yield. The substrate reactivity decreases in order 1,4- 1,3- > 1,2-disubstitution. Prolonged treatment (20 – 50 h) is needed to achieve the conversion to acid or for transformation of the second cyano group.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Changes in mediobasal hypothalamic dopamine and GABA release: A possible mechanism underlying taurine-induced prolactin secretion

Summary Taurine (Tau), a putative inhibitory amino acid neurotransmitter, has been shown to stimulate prolactin (PRL) release. Using ovariectomized, estrogen-replaced adult rats we investigated initially the effect of this amino acid, injected by different routes, on PRL secretion in vivo. Tau (100–500 mg/kg) had no effect on PRL release when given i.p.; 15 min after i.c.v. injection of Tau (3moles), a significant increase in serum PRL levels was observed (78 ± 9 ng/ml over basal levels, p < 0.01 vs. controls). In vitro (cultured anterior pituitary cells) PRL release was not affected by a 5 h incubation with Tau (10^–3–10^–8 M). Basal dopamine (DA) or gamma-aminobutyric acid (GABA) output from superfused mediobasal hypothalamic fragments (MBH) was not affected by Tau (10^–3 M or 10^–5 M). However, during stimulation with KCl (50mM), Tau (10^–3 M) significantly lowered DA release, and increased GABA output. It is concluded that Tau acts at a central level to increase PRL secretion, most probably by modulating the hypothalamic release of neurotransmitters controlling lactotroph function.     

Inhibition of the Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Alkyl-Substituted Diatomic Phenols

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3,5,5-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20°C in 0.015 M phosphate–citrate buffer (pH 6.0) containing organic cosolvents (0–10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (K _i), the duration of the lag period in the oxidation product formation (), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate–diatomic phenol pair. Various substrate–diatomic phenol pairs exhibited K _i values within the range of 11–240 M andfvalues from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine–substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate–substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Hormones andCuscuta development: interaction of cytokinin and indole-3-acetic acid in the growth and curvature of subapical stem segments

Cuscuta stem (vines) exhibits two modes of growth—longitudinal elongation forming free-hanging vines, or coiling growth to twine around the host. The elongation zone of free-hanging vine extended up to 160 mm from the stem apex and in vivo growth rate (during 8 h of growth) was maximal in the 20-to-40-mm region. While gibberellic acid (GA₃) or fusicoccin (FC) could maintain (GA₃) or enhance (FC) the growth rate of apical (10 or 25 mm) segments, indole-3-acetic acid (IAA) (10 M) induced growth only in subapical (5–160 mm) segments. In vitro growth rate induced by IAA (10 M) was similar to the in vivo growth rate up to 40 mm. Thereafter, up to 100 mm, IAA induced growth rate exceeded in vivo growth. p ]Subapical segments (13 mm) from 5- to 40-mm regions responded to a cytokinin (BA, Z, or iP) or to low IAA (0.1 M) with curved growth, whereas the segments grew straight in the presence of high IAA (10 M). Curvature (measured as the angle subtended at the center of the circle of which the segment formed an arc) induced by BA and low (0.1 M) IAA was greater than either added separately. Besides, segments induced to curve in BA + low-IAA solution could be made to straighten out by transferring to a solution containing high IAA (10 M) with or without BA. Thus in vivo patterns of straight and coiling growth could be mimicked reversibly in vitro by adjusting the relative concentrations of cytokinin and auxin; low auxin and cytokinin induced coiling growth, whereas high auxin and cytokinin induced straight growth. p ]Beyond 40 mm, BA had no growth-promoting or curvative-inducing effect.Cuscuta vine segments thus showed sequential sensitivity to applied hormones, the apical region (0–25 mm) to GA₃, the subapical (5–40 mm) region to BA and IAA and the region beyond (40–160 mm) to IAA alone.     

Somatic embryogenesis and plant regeneration in Gymnema sylvestre

Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from hypocotyl, cotyledon and leaf explants excised from seedlings of Gymnema sylvestre. Embryogenic callus was induced on Murashige and Skoog (MS) medium containing 2,4-D (0.5–5.0 M) +BA (0.5–2.0 M) and 2% (w/v) sucrose in 6–8 weeks of culture. Globular/heart stage embryos developed on induction medium. These embryos produced torpedo and cotyledon stage embryos upon sub-culturing on embryo maturation medium EM8 (medium containing MS salts, B5 vitamins, 0.5 M BA and 2% sucrose). Embryo germination and plantlet formation was achieved by sub-culturing mature embryos on fresh EM8 medium. The plantlets were acclimatized in the greenhouse.     

Five new species of coccidia (Apicomplexa: Eimeriidae) from Madagascan chameleons (Sauria: Chamaeleonidae)

Coprological examination of 19 Madagascan chameleons of the genera Furcifer and Brookesia revealed the presence of five new coccidian species. Isospora brygooi n. sp. from Furcifer pardalis has spherical to subspherical oöcysts with a slightly pitted wall, 20.7 (17–24.5) × 19.3 (16–23) m and broadly ellipsoidal sporocysts, 12.2 (11.5–13) × 8.1 (8–8.5) m, with Stieda and substieda bodies. Oöcysts of Eimeria glawi n. sp. from Furcifer pardalis are cylindrical to ellipsoidal, 27.7 (26–29.5) × 18.4 (17–19) m, with ellipsoidal sporocysts, 7.3 (6.5–8) × 5.2 (5–5.5) m. E. vencesi n. sp. described from F. pardalis has spherical to subspherical oöcysts, 14.3 (13–15.5) × 13.0 (12–13) m, with small granules, one to three globular polar granules and ellipsoidal sporocysts, 7.3 (6.5–8) × 5.2 (5–5.5) m. E. worthi n. sp., described from Furcifer oustaleti has spherical oöcysts, 17.9 (17.5–19.0) × 15.0 (14.5–16.0) m without a polar granule and ellipsoidal to cylindroidal sporocysts, 8.2 (7.0–9.5) × 5.8 (5.0–6.5) m. Oöcysts of E. brookesiae n. sp. from Brookesia decaryi are cylindrical, 25.6 (23–27) × 15.0 (13–16) m with ellipsoidal sporocysts, 10.1 (9–11) × 6.9 (6–7) m. Endogenous development of E. vencesi is confined to the intestine, while that of E. glawi occurs in the gall-bladder.     

Acute tolerance to ethanol inhibition of NMDA-induced responses in rat rostral ventrolateral medulla neurons

The present study was performed to examine the effects of acute ethanol exposure on N-methyl-D-aspartate (NMDA)-induced responses and the development of acute tolerance in rat rostral ventrolateral medulla (RVLM) in vivo and in vitro. Repeated microinjections of NMDA (0.14 nmol) into the RVLM every 30 min caused reproducible increases in mean arterial pressure in urethane-anesthetized rats weighing 325–350 g. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA-induced pressor effects in a blood-concentration-dependent and reversible manner. The inhibitory effect of ethanol was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol (0.32 g). A high dose of ethanol (0.32 g) had no significant effects on-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid,-aminobutyric acid and glycine-induced changes in blood pressure. In vitro studies showed that ethanol (10–100 mM) dose-dependently inhibited inward currents elicited by pressure ejection of NMDA (10 mM) in RVLM neurons of neonatal brainstem slice preparations. When the superfusion time of ethanol (100 mM) was increased to 50 min, its inhibitory effect decreased gradually after 30–40 min in 60% of RVLM neurons examined. These data suggested that ethanol inhibition and subsequent tolerance development is associated with changed sensitivity to NMDA in the RVLM, which may play important roles in the ethanol regulation of cardiovascular function.     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

Rapid in vitro Regeneration of Sesbania drummondii

This paper describes rapid propagation of Sesbania drummondii using nodal explants isolated from seedlings and young plants. The nodal segments proliferated into multiple shoots on Murashige and Skoog's (MS) medium supplemented with 22.2 M benzyladenine. MS medium containing 2.2 and 4.5 M thidiazuron induced 5 – 6 shoots per stem node from 3-month-old plants. Nodal explants when cultured on MS medium containing combinations of benzyladenine (8.8 and 11.1 M) and indole-3-butyric acid (0.24 – 2.46 M) or indole-3-acetic acid (0.28 – 2.85 M) gave lesser number of shoots. Callus induced on cotyledonary explants when subcultured on 2.2 M thidiazuron containing medium resulted in its mass proliferation having numerous embryoid-like structures. Indole-3-butyric acid (0.24 – 2.46 M) was found suitable for root induction. In vitro regenerated plants were acclimatized in greenhouse conditions.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Indigogenic methods for glycosidases

Summary In the histochemical detection of -D-glucosidase the indigogenic method of Pearson et al. was tested and evaluated. 4-Cl-5-Br-3-indolyl--D-glucoside was used as the substrate.Intestinal -D-glucosidase is firmly bound to the structure. About 60% of activity survives 2 hours fixation in cold 4% formaldehyde and some activity can be demonstrated even in paraffin sections after a shortened embedding.The localization obtained with the original method is not correct. Due to a slow oxidation of indoxyl in the acid pH range and to hydrogen peroxide formation indigo is deposited in sites with an active peroxidase or pseudoperoxidase. The addition of horse-radish peroxidase improves the localization but does not entirely prevent diffusion artifacts. A striking improvement of the localization can be achieved by a mixture of ferri-ferrocyanide. 3.1 · 10^–3 M concentration of this oxidation catalyst which is still very effective causes only a 26% inhibition of the enzyme activity as revealed by biochemical assays in homogenates of rat intestine.A new medium was devised consisting of 0.1 M citrate phosphate buffer pH 5.5, 8 · 10^–4 M 4-Cl-5-Br-3-indolyl--D-glucoside and 3.1 · 10^–3 M ferri-ferrocyanide mixture. With this medium a very clear brush border localization of the enzyme activity (activities) in enterocytes of the rat and human intestine was demonstrated. This activity is present in differentiated enterocytes covering the villi. The highest activity resides in the jejunum. Enzyme activity is considerably inhibited by galactonolactone (5 · 10^–3 M) and gluconolactone (4 · 10^–4 M). It is completely inhibited by florizin (5 · 10^–3 M). Cellobiose (8 · 10^–2 M) caused 65%, lactose (8 · 10^–2 M) 48% and glucose (8 · 10^–2 M) 35% inhibition (data were obtained by cytospectrophotometry). In patients with celiac sprue the activity is very much decreased. Its restitution after a gluten-free diet proceeds roughly parallel to that of lactase. The relationship of the demonstrated activity (activities) to florizin hydrolase and lactase is discussed.In the kidney the reaction is very weak and is confined to the cells of proximal convoluted tubules (diffuse staining with some enhancement at the luminal surface of the brush border). The method is also very useful for processing zymograms.