Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.     

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.     

Vascularization and related distribution of leucocytes in carp, Cyprinus carpio L., head kidney

The distribution of lymphoid cells in the carp head kidney was investigated in relation to the vascular system. Blood vessels in the head kidney were histologically identified into arteries, sinusoids and two types of veins: renal veins and portal, which were distinguished by India ink injection into the caudal vein. By histological and histoplanimetrical observations it was found that the head kidney contained a number of lymphoid cells, which mainly aggregate around the connections between the portal veins and sinusoids, and that the cellular density of the aggregations was higher than in the thymus.
Pigment-containing cell clusters were also observed around these connections. This arrangement of the blood vessels suggests that it is one of the structures able to trap foreign materials, and the occurrence of the lymphoid clusters around the portal veins is a phylogenetic sign of the morphological division between granulopoietic and lymphatic tissues in the carp head kidney.     

A murine very late activation antigen-like extracellular matrix receptor involved in CD2- and lymphocyte function-associated antigen-1-independent killer-target cell interaction

CD2 and lymphocyte function-associated antigen (LFA)-1 are well known as T cell adhesion molecules involved in killer-target cell interactions. However, our recent study revealed that molecule(s) other than CD2 and LFA-1 might be involved in the lymphokine-activated killer (LAK) cell cytotoxicity against certain target cells. In order to characterize such unknown molecules, we established a mAb (RMV-7) which could inhibit CD2/LFA-1-independent LAK cell cytotoxicity and binding to target cells at the effector site. The Ag identified by RMV-7 appeared on splenic T cells late after mitogenic stimulation and was a noncovalently linked heterodimer composed of a 140-kDa alpha-chain and a 95-kDa beta-chain. RMV-7 blocked LAK cell binding to fibronectin (FN), fibrinogen, and vitronectin but not that to laminin or type IV collagen, indicating that the RMV-7-defined molecule is a unique extracellular matrix receptor for FN, fibrinogen, and vitronectin. One of its ligand, FN, was found on the surface of several target cells, and LAK cell cytotoxicity against them was blocked by anti-FN antibody at the target site. Similarly, cytotoxicity of a H-2d-specific CTL clone was inhibited by RMV-7 and anti-FN antibody as well. These results indicate that a unique very late activation Ag-like extracellular matrix receptor on murine CTL and LAK cells contributes to target cell binding and cytotoxicity.     

Incidence of Clostridium botulinum in honey of various origins

By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one. Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C. Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F). The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%). It was estimated that most positive samples contained spores in one per gram or lower concentrations. One sample contained 4 type A spores per gram and another 36-60 type F spores per gram. No distinct biochemical properties were found with the honey isolates.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.     

The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes. cDNA clones for PMP70 were isolated and sequenced. The predicted amino acid sequence (659 amino acid residues) revealed that the carboxyl-terminal region of PMP70 has strong sequence similarities to a group of ATP-binding proteins such as MalK and Mdr. These proteins form a superfamily and are involved in various biological processes including membrane transport. Limited protease treatment of peroxisomes showed that the ATP-binding domain of PMP70 is exposed to the cytosol. The hydropathy profile, in comparison with those of several other members of the ATP-binding protein superfamily, suggests that PMP70 is a transmembrane protein possibly forming a channel. Based on these results, we propose that PMP70 is involved in active transport across the peroxisomal membrane.     

Possible mechanism of gastric mucosal protection by epidermal growth factor in rats

The mechanism of the protection by human epidermal growth factor (hEGF) against the gastric mucosal lesions induced by acidified ethanol was studied in rats. At different times following the subcutaneous administration of hEGF (30 micrograms/kg), intragastric acidified ethanol (EtOH: 0.125 M HC1 = 50:50 v/v%) was administered to induce an experimental gastric mucosal lesion. Mean length of the lesion in the gastric mucosa was used as a lesion index. Extravasation of intravenously injected Evans blue into the gastric wall and gastric contents was used as an indicator of vascular permeability. Pretreatment with hEGF decreased both the gastric mucosal lesions and the increase of vascular permeability caused by acidified ethanol with similar time profiles relative to pretreatment with hEGF. Maximal protective actions of hEGF occurred about 10 to 30 min after the observed peak plasma concentration of hEGF. Indomethacin and N-ethylmaleimide, but not iodoacetamide, blocked the protective action of hEGF, indicating that endogenous prostaglandins and/or sulfhydryls may participate in the protective action of hEGF. The content of endogenous nonprotein sulfhydryls in the gastric mucosa decreased markedly after acidified ethanol. However, pretreated hEGF did not restore the sulfhydryl contents. Thus, it seemed that endogenous prostaglandins, but not sulfhydryls, are the probable mediators for protection against gastric mucosal injury caused by acidified ethanol.     

Purification of a Dithiothreitol-Sensitive Tetrameric Protease from Spinach PS II Membranes

A protease with a tetrameric quaternary structure was extractedwith 1 M NaCl from spinach PS II membranes and purified by hydrophobic,anion-exchange and gel-filtration chromatography using onlybuffers of high ionic strength. Gel-filtration chromatographyresulted in elution of the protease in fractions that correspondedto molecular masses of 156 kDa and 39 kDa, and re-chromatographyof either peak gave both peaks again. This result indicatesthat the protease is represented by an equilibrium between a156-kDa tetramer and a 39-kDa monomer. SDS-polyacrylamide gelelectrophoresis of the protease fractions revealed a polypeptidewhose molecular mass was 39 kDa without prior reduction, butthe molecular mass increased to 41 kDa after prior reductionwith dithiothreitol. This finding suggests that the monomerpossesses an intramolecular disulfide linkage whose reductioncauses a configurational change that increases the effectivemolecular size. The protease had maximum activity at pH 7.0–9.0.The activity was diminished by the presence of 5 mM NaCl andwas almost completely inhibited by 50 mM NaCl. These observationssuggest that an environment of low ionic strength is a prerequisitefor the activity of this enzyme. The protease was inhibitedby dithiothreitol, a result that indicates that the 39-kDa formmaintained by the disulfide linkage is essential for activity.Studies with protease inhibitors suggested that this enzymeis not a serine-protease. ¹Present address: Department of Biomolecular Science, Facultyof Science, Toho University, Miyama 2-2-1, Funabashi, 274 Japan (Received October 19, 1989; Accepted April 12, 1990)