Studies on lipase from Mucor javanicus. I. Purification and properties.

1. Lipase produced by a mold, Mucor javanicus, was purified about 180-fold from the ethanol precipitate of the culture filtrate. Purification was achieved by acid precipitation followed by gel filtrations on Sephadex G-200 (at low ionic strength) and Sephadex G-75 (at a high ionic strength). The purified enzyme preparation showed unusual behavior on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 21 000. The enzyme had a positional specificity towards the position 1 and 3 of triacylglycerols. 2. Lipase in the crude preparation takes an aggregated form. aggregated form was achieved by raising the ionic strength of the medium. 3. The purified lipase preparation from Mucor javanicus exhibits phospholipase A1 activity, hydrolyzing the carboxyl ester at the 1-position of phosphatidylcholine. This activity seems to be due to the action of the lipase itself and not due to any other specific phospholipases.

Role of chronic Pseudomonas aeruginosa infection in airway mucosal permeability.

The role of Pseudomonas aeruginosa infection in airway mucosal permeability was studied in 16 patients with chronic bronchitis by measuring the amounts of radiolabeled albumin in sputum. One group (A) consisted of six patients (two female, four male, 53 +/- 6 years, SEM) with chronic P aeruginosa infection for 5 +/- 0.9 years. Another group (B) consisted of ten patients (five female, five male, 67 +/- 4 years) without P aeruginosa infection for at least two years. No significant differences between groups A and B were found in the volume of sputum (63 +/- 21 ml/day in group A and 45 +/- 8 ml/day in group B, p = 0.44), the obstructive changes (FEV1 of 57 +/- 6 percent in group A and 51 +/- 4 percent in group B), or the duration of disease (19 +/- 4 years in group A and 14 +/- 4 years in group B). Saliva, sputum, and serum samples were collected at intervals of 2 h over an 8-h period, and at 24 h after intravenous administration of 131I-labeled human albumin. For counting, free 131I was removed by dialysis. Radiocounts (cpm) of saliva were significantly smaller than those of sputum or serum. The cpm from each sputum sample was divided by serum cpm at the time of each sampling. Group A showed significantly higher values in the ratio of sputum- to serum-cpm than did group B at all sampling times. Furthermore, the ratios at 2 and 4 h after 131I-albumin injection significantly correlated with sputum volume per day, whereas they did not correlate with any other factors (age, obstructive impairment, and duration of disease). These findings suggest that chronic P aeruginosa infection produces an increase in airway mucosal permeability to albumin.

A novel missense mutation in the early growth response 2 gene associated with late-onset Charcot--Marie--Tooth disease type 1.

A novel mutation (Arg381Cys) in the second zinc-finger domain of early growth response 2 (EGR2) was identified in a late-onset Charcot--Marie--Tooth disease type 1 (CMT1) patient. This patient had initial symptoms of numbness and weakness in the leg at age 59, and a median nerve motor conduction velocity of 27 m/s. A sural nerve biopsy showed a severe loss of myelinated fibers with numerous onion bulbs. This is the first report of the EGR2 mutation presenting a late onset of CMT1 phenotype. Its mutation was a different amino acid substitution at codon 381 (Arg381His) which demonstrated congenital hypomyelinating neuropathy or early-onset CMT1. This report suggests that the EGR2 mutation represents divergent phenotypes at codon 381, which may be a mutation hotspot.

The measurement of physiological tooth displacement in function.

The purpose of this work was to investigate the physiological tooth displacement in function. We developed the tooth displacement transducer Type M-3 and measured the three dimensional displacement of the upper and lower left first molars in function. The summary is as follows: 1. Magnesensors were used in the tooth displacement transducer Type M-3. Its total weight was 48.7 g. and the measuring pressure was kept less than 0.4 g. It was fixed at the labial surface of the anterior teeth using a paraocclusal splint. 2. The linearity error of the transducer was less than 2% within a range of +/-200 microm. 3. The upper left first molar showed displacement in the palatal and apical direction during clenching at the intercuspal position with habitual occlusal force or during the biting of articulating papers. The displacement varied between 40 and 136 microm. 4. The lower left first molar showed displacement in the apical and lingual direction during clenching at the intercuspal position or during the biting of articulating papers. The displacement varied between 29 and 65 microm. 5. The upper left first molar showed displacement in the palatal and apical direction during mastication. The displacement varied between 68 microm and 79 microm. The upper teeth moved first in the apical direction, then moved toward the labial direction. 6. In the lingual direction, the lower left first molar showed displacement ranging from 11 to 71 microm and 17 to 39 microm in the buccal direction during mastication.

[A case of cephalic tetanus presenting with opisthotonus].

We report a case of cephalic tetanus presenting with opisthotonus. A 49-year-old man was admitted because of repeated convulsions. The patient was a garbage truck driver known to be alcoholic, who fell down to suffer an injury of the left face two days before the onset of convulsion. Intravenous administration of diazepam and phenytoin partially relieved the convulsions. Anti-tetanus human immuno-globulin was also administered despite absence of typical clinical sign. Six hours later, however, the patient became unable to open the mouth, i.e. lockjaw developed, and the diagnosis of tetanus was made. Additional anti-tetanus human immunoglobulin of 3,000 units and 4,500 units on the next day rapidly relieved the lockjaw, convulsion, and general muscle rigidity without sequalae. The patient showed transient bilateral facial palsy and rotatory nystagmus during the course. Cephalic tetaus is characterized by a history of an injury of the head and a short latency before developing generalized tonic convulsion or opisthotonus. While a typical case presents with lockjaw, our case presented with opisthotonus, presumably because of early systemic lymphatic spreading of tetanus toxin. Early diagnosis and treatment is important to prevent generalized convulsions which are more frequent and sometimes lethal in cephalic tetanus than the common form.

[Anesthetic management of a patient with Sjögren's syndrome and pulmonary fibrosis].

We described an anesthetic management of a 63 year old man complicated with Sjögren's syndrome and pulmonary fibrosis. Sjögren's syndrome is characterized by pathological dryness of the mouth, cornea and other exocrine glands. Anesthesia was induced with thiopental and the trachea was intubated smoothly following intravenous succinylcholine. Anesthesia was maintained with enflurane in nitrous oxide and oxygen. Intraoperative muscle relaxation was adequately obtained by pancuronium. Before and during anesthesia, atropine and other drying agents were avoided. Physiological saline solution was instilled on the eyes every ten minutes against dryness. A heat and moisture exchanger (Humi-Vent) was used to maintain high moisture of the respiratory tract. Anesthesia was carried out successfully.

[Difficult tracheal intubation and abnormal response to thiopental in a patient with arthrogryposis multiplex congenita].

Anesthesia was administered for six times to a patient with arthrogryposis multiplex congenita (AMC) at the age of 10 to 17 years. In the first four occasions of anesthesia, difficult tracheal intubation was encountered due to limited neck extension, inadequate mouth opening and the short epiglottis. On the fifth anesthesia, the patient remained conscious even after intravenous injection of thiopental 6.6 mg.kg-1, and enflurane in combination with nitrous oxide was administered to induce anesthesia. During the induction of the sixth anesthesia, excessive oral secretion and severe continued nausea were observed just after intravenous administration of thiopental 5.3 mg.kg-1. These were overcome by intravenous administration of ketamine 2.0 mg.kg-1. Problems such as difficulty in tracheal intubation and abnormal response to thiopental need special attention in patients complicated with AMC, particularly during induction of anesthesia.

[Influence of occlusal contacts on tooth displacement].

During function, the tooth displacement path depends on occlusal contacts of the upper and lower teeth. Therefore, we must reconstruct occlusal relation carefully so as not to disturb physiological tooth displacement. In this investigation, tooth displacements were measured with the Displacement Transducer type M-3 in varying occlusal contact. The following conclusions were obtained. 1. The location of occlusal contacts influenced the tooth displacement of the upper first molar. Especially, an occlusal contact on the lingual inclination of the lingual cusp, buccal cusp had great influence. The tooth showed displacement toward the buccal direction, and it differed from the physiological tooth displacement path during clenching at the intercuspal position. 2. The location of occlusal contacts had little influence on the tooth displacement of the lower first molar. The tooth showed displacement toward the lingual direction. It was similar to the physiological tooth displacement. 3. On the upper first molar, the location of occlusal force influenced the Stress-Strain curve. Especially, the occlusal force on the buccal cusp had great influence on the amount of tooth displacement. 4. On the lower first molar, the location of occlusal force influenced the Stress-Strain curve. Especially, the occlusal force on the lingual cusp brought much displacement and a 2-phase displacement path, at times. Therefore, occlusal contacts have close relation to tooth displacement, and any occlusal reconstruction should harmonize with tooth displacement representing periodontal tissue distortion.

Electroblotting of double-stranded DNA for hybridization experiments: DNA transfer is complete within 10 minutes after pulsed-field gel electrophoresis.

A rapid, simple, and efficient method for DNA blotting is presented. This method is characteristic in that DNA is transferred without denaturation from an electrophoretic gel to a membrane in low-salt concentrations and denatured on the membrane after blotting. More than 89% of double-stranded DNAs ranging in size from 75 to 1.9 million bp can simultaneously be transferred from the gel to a positively charged nylon membrane within 10 min. The present "low-salt electroblotting" method is superior to other blotting methods in that it saves time and labor and its high and even transfer efficiency makes it useful for hybridization analysis.

Synthesis and application of neoglycolipids for liposome modification.

We synthesized various glycolipid derivatives and examined the in vivo behaviors of liposomes modified with these novel glycolipid derivatives. Gal-t-psa (1,¿8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl¿-beta-D- galactoside), Lac-t-psa (3, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl beta-D-lactoside) and GalNAc-t-psa (4, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl 2-acetamido-beta-D-galactopyranoside) modified liposomes were recognized by the liver. Lac-t-psa (3) modified liposome was accumulated to the highest degree, followed by GalNAc-t-psa (4) modified liposome and then Gal-t-psa (1) modified liposome. The intrahepatic distributions of Gal-t-psa (1), GalNAc-t-psa (4), Glc-t-psa (2, 8-(2-hexadecyloctadecanoylamido)-3,6-dioxaoctyl beta-D-glucopyranoside) and Lac-t-psa (3) modified liposomes were investigated. GalNAc-t-psa (4) and Lac-t-psa (3) modified liposome were accumulated to greater extents than Gal-t-psa (1) modified liposome in hepatic parenchymal cells. The intrahepatic distribution of these liposomes showed that Lac-t-psa (3) and GalNAc-t-psa (4) were preferable to Gal-t-psa (1) for the selective delivery of liposomes to hepatic parenchymal cells.

Proteoglycan synthesis in the intervertebral disk nucleus: the role of extracellular osmolality.

Proteoglycans, through their polyelectrolyte properties, regulate the ionic composition and hence the osmotic pressure of the extracellular matrix. We measured the change in [35S]sulfate incorporation, a marker of proteoglycan synthesis, in explants of bovine nucleus pulposus. During incubation, nucleus slices swelled 200% and proteoglycans leached from the matrix, so that extracellular osmolality fell from 420-450 to approximately 300 mosmol/kg H2O. When in vivo extracellular osmolality was maintained either by adding 80 mM NaCl or 150 mM sucrose to the swollen tissue or by preventing swelling, synthesis rates were 260-280% greater than in swollen tissue. Synthesis rates also increased 200% in cells isolated from the nucleus pulposus by enzyme digestion when medium osmolality was raised from 280 to 430 mosmol/ kgH2O by sucrose addition. The cells, either in the tissue or isolated from it, swelled by more than 20% as osmolality fell from 430 to 280 mosmol/kgH2O and showed little regulatory volume decrease over 150 min. Synthesis rates thus appear to be regulated by extracellular osmolality rather than by the macromolecular composition of the matrix and correlated well with measured changes in cell volume.

Massive actin polymerization induced by phosphatidylinositol-4-phosphate 5-kinase in vivo.

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K Ialpha induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.

Hepatic accumulation of glutamic acid branched neogalactosyllipid modified liposomes.

We synthesized branched type galactosyllipid derivatives for liposome modification for the targeting of asialoglycoprotein receptors on the surface of liver cells. Galactose was coupled to the alpha- and gamma-carboxyl groups of glutamic acid via a triethyleneglycol spacer, then this glutamic moiety was bound to the lipid anchor. Ricinus communis agglutinin (RCA120) induced the agglutination of liposomes modified with mono-, bi- and tri-antennary neogalactosyllipid. With the bi- or tri-antennary derivatives, agglutination was observed at fewer galactosyl residues on the liposomes. We examined the effect of the branching structure in vivo. The difference in accumulation of liposomes between non-branched type neogalactosyllipid and branched type neogalactosyllipid was not large. Liver accumulation of liposomes depended on the galactosyl residues. The number of galactosyl residues was more effective for accumulation in the liver than for branching. We studied the effect of asialofetuin preinjection on the hepatic accumulation of neogalactosyllipid modified liposomes. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. The effect of preinjection was almost equal among the ligands. These results show that the saccharide density on the liposome surface seemed to be a more important factor than the branching structure of the ligand for liver targeting.

Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes.

The selective delivery of human recombinant interferon (IFN)-gamma to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-gamma was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [32P]IFN-gamma was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [32P]IFN-gamma were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4 degrees C) was one-fifth of the uptake (estimated at 37 degrees C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-gamma into hepatocytes in vitro.

Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-gamma by human hepatoma cells and its inhibitory effect on hepatitis B virus replication.

Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N-liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes.

Interaction of recombinant human interferon-gamma with liposomes.

The interaction of recombinant human interferon-gamma (IFN) with egg phosphatidylcholine liposomes was studied. IFN which binds to liposomes was dependent on the liposomal charge and pH, and a preferential binding was observed in negatively charged liposomes at pH 7.4-10. Electron-microscopic observation showed that the increased liposomal turbidity induced by IFN was due to liposomal aggregation, and the increased turbidity could be decreased by the addition of NaCl. Thus, ionic binding may participate in this interaction. But, when the incubation time was longer, the liposomal aggregation was not decreased by the addition of NaCl, and the leakage of the entrapped marker, calcein, was observed. Electron-microscopic analysis showed that this leakage resulted from the morphological change of liposomes. From these findings, ionic binding may participate in the interaction between IFN and liposomes and then develop a morphological change in negatively charged liposomes under the neutral pH condition.

Anterior interbody fusion with and without interspinous block implementation for lumbar isthmic spondylolisthesis.

The clinical and radiographic results of lumbar isthmic spondylolisthesis were compared between interspinous block-assisted anterior interbody fusion (block-assist group, n = 16) and anterior interbody fusion with no use of the block (nonassist group, n = 17) with an average follow-up of 7 years (range 1 1/3-13 years). Satisfactory relief of low-back pain, significantly early interbody union (union rate 88%, p < 0.05), and spontaneous fusion of pars defect (fusion rate 44%, p < 0.05) were obtained in the block-assist group. In the nonassist group, interbody union was markedly delayed (p < 0.05), the union rate was 53%, and spontaneous fusion of the pars was found in 12% of patients. The overall clinical results at final follow-up in both groups did not show a statistically significant difference, but the results tended to be superior in the block-assist group.

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum.

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.