Solution structure and activity of mouse lysozyme M

The three-dimensional structure of mouse lysozyme M, glycoside hydrolase, with 130 amino acids has been determined by heteronuclear NMR spectroscopy. We found that mouse lysozyme M had four alpha-helices, two 3(10)helices, and a double- and a triple-stranded anti-parallel beta-sheet, and its structure was very similar to that of hen lysozyme in solution and in the crystalline state. The pH activity profile of p-nitrophenyl penta N-acetyl-beta-D-chitopentaoside hydrolysis by mouse lysozyme M was similar to that of hen lysozyme, but the hydrolytic activity of mouse lysozyme M was lower. From analyses of binding affinities of lysozymes to a substrate analogue and internal motions of lysozymes, we suggest that the lower activity of mouse lysozyme M was due to the larger dissociation constant of its enzyme-substrate complex and the restricted internal backbone motions in the molecule.     

Extended blood half-life of monomethoxypolyethylene glycol-conjugated hen lysozyme is a key parameter controlling immunological tolerogenicity

The blood half-life of a protein is prolonged by conjugating a protein with a linear amphiphilic polymer, monomethoxypolyethylene glycol (mPEG). The conjugation gives a protein immunotolerogenicity; hence, it is likely that the long half-life is crucial for the tolerogenicity. We prepared a tolerogenic mPEG conjugate of hen egg lysozyme (mPEG_1.5-HEL), which is conjugated 1.5-fold the molecular weight of mPEG against that of HEL, and evaluated the relationship between in vivo stability and the tolerogenicity. mPEG_1.5-HEL retained immunogenicity to prime HEL-specific T cell and antibody responses and had a long blood half-life, more than 27 times that of native HEL. The tolerant state was maintained as long as mPEG_1.5-HEL was detected in sera. With a decrease in the blood mPEG_1.5-HEL level, the tolerant state returned gradually to the responsive state; however, reinjection of mPEG_1.5-HEL again restored the tolerance. Thus, the extended blood half-life of HEL by mPEG conjugation is probably vital for establishing and maintaining the tolerant states. Received 17 May 1999; accepted 1 June 1999     

Periplasmic lysozyme inhibitor contributes to lysozyme resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The product of the Escherichia coli ORFan gene ykfE was recently shown to be a strong inhibitor of C-type lysozyme in vitro. The gene was correspondingly renamed ivy (inhibitor of vertebrate lysozyme), but its biological function in E. coli remains unknown. In this work, we investigated the role of Ivy in the resistance of E. coli to the bactericidal effect of lysozyme in the presence of outer-membrane-permeabilizing treatments. Both in the presence of lactoferrin (3.0 mg/ml) and under high hydrostatic pressure (250 MPa), the lysozyme resistance of E. coli MG1655 was decreased by knock-out of Ivy, and increased by overexpression of Ivy. However, knock-out of Ivy did not increase the lysozyme sensitivity of an E. coli MG1655 mutant previously described to be resistant to lysozyme under high pressure. These results indicate that Ivy is one of several factors that affect lysozyme resistance in E. coli, and suggest a possible function for Ivy as a host interaction factor in commensal and pathogenic E. coli.Received 12 February 2004; received after revision 11 March 2004; accepted 16 March 2004     

Lysozyme activity in female genital tissues of normal and genetically lysozyme-deficient rabbits

The internal genitalia of female normal rabbits and mutant lysozyme-deficient rabbits, which lack genetically the leukocytic isozyme of lysozyme, were assayed for lysozyme activity. The ovaries, uteri, and vaginas of the lysozyme-deficient rabbits had less than 20% of the lysozyme activity of normals. The oviducts, and in particular the caudal portions of the oviducts, had lysozyme activities up to 71% of the levels in normals. These observations suggest that the lysozymes of oviduct and leukocytes of rabbits are under the control of different genes.     

Lysozyme activity in female genital tissues of normal and genetically lysozyme-deficient rabbits

Summary The internal genitalia of female normal rabbits and mutant lysozyme-deficient rabbits, which lack genetically the leukocytic isozyme of lysozyme, were assayed for lysozyme activity. The ovaries, uteri, and vaginas of the lysozyme-deficient rabbits had less than 20% of the lysozyme activity of normals. The oviducts, and in particular the caudal portions of the oviducts, had lysozyme activities up to 71% of the levels in normal. These observations suggest that the lysozymes of oviduct and leukocytes of rabbits are under the control of different genes.Supported in part by grants RR00515, RR05465, K04-RR00003, and T32-GM07160 from the National Institutes of Health. We acknowledge the excellent technical assistance of LeeAndra Froseth.     

Malignant hyperthermia: Molecular defects in membrane permeability

Summary Malignant hyperthermia (MH), a genetically inherited disorder of skeletal muscle, is due to molecular defect in membrane permeability. The alteration in membrane permeability is suggested to be due to enhanced phospholipase A₂ activity which is responsible for the increased level in sarcoplasmic Ca²⁺. The excess Ca²⁺ is responsible for muscle hyper-rigidity and enhanced rate of glycolysis, resulting in a rapid rate of lactic acid production and a low pH in MH muscle.     

Cationic and secretory effects of BPDZ 44 and diazoxide in rat pancreatic islets

The present study aimed at comparing the effects of low concentrations of BPDZ 44, a new pyridothiadiazine derivative, and diazoxide on⁸⁶Rb outflow,⁴⁵Ca outflow,⁴⁵Ca uptake and insulin release from rat pancreatic islets. Both drugs caused similar modifications, but the effects of BPDZ 44 on the cationic and secretory events were much more marked than those of diazoxide. It is suggested that BPDZ 44 could be valuable tool for further studies of the K_ATP channels.     

Chromate reduction inStreptomyces

Summary Streptomyces species 3M grew in peptone yeast extract medium with 1000 g/ml K₂Cr₂O₇. Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr⁶⁺ in the reaction mixture. The level of Cr⁶⁺ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000×g for 1 h, in the presence of NADH. The reducing enzyme was associated with this cell fraction. The enzyme was constitutive and reduced Cr⁶⁺ to Cr³⁺.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O₂ uptake and generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H₂O₂ production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.Acknowledgments. This work was aided by grants from the University of Buenos Aires, the Scientific Office of the American States Organization and CEDIQUIFA (Buenos Aires). S.G.G. and M.P.M.P. are Research Fellows and A.O.M.S. is Career Investigator of CONICET (Argentina). L. T. Viñas and M. G. Gutierrez lent able technical assistance.     

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

Selective pharmacological Na⁺/H⁺ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit ²²Na⁺ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na⁺-dependent (NHE3-sensitive) component of apical dipeptide ([¹⁴C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical ²²Na⁺ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [¹⁴C]Gly-Sar uptake in the absence of Na⁺ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H⁺ electrochemical gradient). EIPA (at 100 μM) has similar effects, but at higher concentrations (>200 μM) also has direct inhibitory effects on hPepT1.Received 28 February 2005; received after revision 20 April 2005; accepted 20 May 2005     

Sterol binding by OSBP-related protein 1L regulates late endosome motility and function

ORP1L is an oxysterol binding homologue that regulates late endosome (LE) positioning. We show that ORP1L binds several oxysterols and cholesterol, and characterize a mutant, ORP1L Δ560–563, defective in oxysterol binding. While wild-type ORP1L clusters LE, ORP1L Δ560–563 induces LE scattering, which is reversed by disruption of the endoplasmic reticulum (ER) targeting FFAT motif, suggesting that it is due to enhanced LE–ER interactions. Endosome motility is reduced upon overexpression of ORP1L. Both wild-type ORP1L and the Δ560–563 mutant induce the recruitment of both dynactin and kinesin-2 on LE. Most of the LE decorated by overexpressed ORP1L fail to accept endocytosed dextran or EGF, and the transfected cells display defective degradation of internalized EGF. ORP1L silencing in macrophage foam cells enhances endosome motility and results in inhibition of [³H]cholesterol efflux to apolipoprotein A-I. These data demonstrate that LE motility and functions in both protein and lipid transport are regulated by ORP1L.     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

Dynamic laser light scattering studies of the effects of pyrophosphate on cyclic motions of cross-bridges in isolated thick myofilaments fromLimulus striated muscle

Pyrophosphate (PP_i) is a non-hydrolyzable ATP analogue known to affect the binding between myosin heads and actin. By using a dynamic laser light scattering method, we have shown that 1–2 mM PP_i enhances the increase in values induced by Ca²⁺ in isolated thick myofilaments fromLimulus striated muscle. However, similar treatment has no effect on the values of filaments suspended in either relaxing solution or ATP-free solution. is the average linewidth of the photoelectron count autocorrelation function of the light scattered. PP_i had no effect on the increase of values by Sr²⁺ but it substantially increased the values of the thick myofilaments suspended in Ba²⁺-substituted Ca²⁺ activating solution. The results show that PP_i also affects the energy-requiring cyclic cross-bridge motions.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

Uninterrupted protein synthesis is essential for survival in the early stages of carbontetrachloride-induced hepatocellular necrosis in the mouse

Summary The fatal syndrome produced by cycloheximide given 6 h after a hepatonecrogenic dose of CCl₄ is due neither to direct toxic synergism between CCl₄ and cycloheximide nor to transient sinusoidal thrombosis. It is suggested that survival in the presence of unknown factors released from dying liver cells requires uninterrupted protein synthesis. The life-saving effect of sterilization of the intestine by antibiotics indicates that the gut flora or its products play a vital role in pathogenesis.Acknowledgments. I wish to thank Mr C. R. West for carrying out the statistical analysis, Mrs Brenda Brooks for histological processing, and Berk Pharmaceuticals Ltd for information on Ancrod defibrination in mice.     

Dissection of cytotoxic and helper T cell responses

Cytotoxic (CD8⁺) and helper (CD4⁺) T cells play a crucial role in resolving infections by intracellular pathogens. The development of technologies to visualize antigen-specific T cell responses in mice and men over the past decade has allowed a dissection of the formation of adaptive T cell immunity. This review gives a brief overview of the currently used detection techniques and possible future additions. Furthermore, we discuss our current understanding of the formation of antigen-specific T cell responses, with particular attention to the similarities and differences in CD4⁺ and CD8⁺ T cell responses, the functional heterogeneity within responder T cell pools and the regulation of CD8⁺ T cell responses by dendritic cells and CD4⁺ helper T cells. Received 16 June 2005; received after revision 2 August 2005; accepted 15 August 2005     

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of α1-adrenergic and σ1 receptors

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of ₁-adrenergic and ₁ receptors, but in the hippocampus it might be dependent on ₁ receptors only. The activation of ₁-adrenergic receptors synergized ₁ receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.Received 7 January 2005; received after revision 19 February 2005; accepted 22 February 2005 Available online 29 March 2005     

The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.