Adipose tissue extracellular matrix: newly organized by adipocytes during differentiation.

The distribution of eight types of extracellular matrix (ECM) proteins (type I-VI) collagen, laminin and fibronectin) in the skeletal muscle of Japanese Black cattle was determined by indirect immunofluorescence using specific antibodies against each protein. ECM proteins were well organized in the intramuscular connective tissue: type I, II, III collagen and fibronectin were localized primarily in the perimysium, type V and VI collagen in both the perimysium and endomysium, and type IV collagen and laminin were virtually confined to the endomysium. In the loose connective tissue holding the adipocytes together to form a tissue mass between the muscular bundles, seven of the ECM proteins not type II collagen were relatively abundant in a disordered arrangement. Further analysis by in vitro immunocytochemical staining also demonstrated that a stromal-vascular preadipocyte cell line (BIP cell), derived from Japanese Black cattle, synthesized various ECMs in much the same way as fibroblasts. Exponentially growing BIP cells with a fibroblastic phenotype were found to produce type II, V, and VI collagens, in addition to the other previously identified connective tissue glycoproteins of mouse 3T3 preadipocytes. When confluent preadipocyte cultures were stimulated with adipogenic medium, a fibrillar network of ECM was observed to bridge the intercellular space and connect adjacent cell surfaces. During adipocyte differentiation, type III collagen and laminin were arranged in a non-fibrous structure, and type-II collagen was only barely detected. These results are supported by the staining of the adipose tissue, where all ECM proteins studied except type II collagen were stained intensely. These data indicate that in vivo under conditions permissive for adipose conversion, the production and organization of ECM, accompanied by hyperplasia and hypertrophy of precursor cells, gives rise to adipose tissue in skeletal muscle with its own ECM products. These data further suggest that each ECM protein might have some role for the adipocytes in forming tissue.

Compact superconducting ring at ritsumeikan university.

A compact superconducting storage ring (0.575 GeV and 300 mA) with a racetrack microtron injection system was installed at Ritsumeikan University and has been operated successfully since April 1996. As the radius of the circular electron orbit is small (0.5 m), it is possible to use a radiation beam of relatively high photon flux at a short distance from the source point. Beamlines have been constructed including those for XAFS, soft X-ray spectroscopy, VUV spectroscopy, fluorescent X-ray analysis, soft X-ray microscopy, X-ray diffraction/scattering and photoelectron spectroscopy; the photoelectron spectrometer is combined with an ion-beam-scattering spectrometer to obtain information both on the electronic states and on the atomic arrangements of a solid surface. In addition there are two beamlines, one for LIGA exposure and the other for synchrotron radiation ablation, which are devoted to producing new devices and materials. The synchrotron radiation facility is open not only to users in the University but also to researchers in industry, governmental institutions and other universities.

Non-destructive, ultrasonic evaluation of meat quality in live Japanese Black steers from coloured images produced by a new ultrasound scanner.

An improved colour scanning scope was used for evaluating meat quality (marbling) of live Japanese Black steers. This equipment consisted of a small size ultrasonic probe (2 MHZ) and LCD display. Seventeen fattened Japanese Black cattle were scanned at the region of the 7th rib about one week before slaughter. A picture of the cross-sectional area of the back was obtained immediately after applying the probe and contained 15 colours representing different signal strengths. The time for each scan was 2 seconds. The picture signals were fed into a computer for rapid estimation of fat percentage of the M. longissimus thoracis. After slaughter, the fat content and chemical characteristics were determined on the M. longissimus thoracis obtained from the same rib section. The range of fat content was 7.0 to 23.7% (average 18.47%). A high correlation coefficient (r = 0.90; r.s.d. = 2.01%) was obtained between actual fat percentage of the M. longissimus thoracis and colour-scanning scope SR200 estimates based on the percentage of the weak blue dot(1) in the echo. Estimates of the subcutaneous fat thickness and the cross-sectional area of M. longissimus thoracis from the scans were in good agreement with the actual carcass measurements (r = 0.69; r.s.d=0.52 cm and r = 0.81; r.s.d. = 4.26 cm(2), respectively). These results show that the new colour scanning scope is a useful instrument for estimating meat quality (marbling) in live cattle.

A preadipocyte clonal line from bovine intramuscular adipose tissue: nonexpression of GLUT-4 protein during adipocyte differentiation.

A clonal bovine intramuscular preadipocyte (BIP) line has been established from the intramuscular white adipose tissue of the M. longissimus thoracis in each of three Japanese Black cattle. Exponentially growing BIP cells exhibited a fibroblastic appearance. Adipocyte differentiation was initiated by treating confluent BIP cells with differentiation medium containing insulin and dexamethasone. Small lipid droplets appeared 5-6 days after stimulation and occupied a large fraction of the cell volume at 10 days and beyond. During the adipose conversion, the incorporation of acetate to the cells gradually increased by 10-fold and reached a maximum at day 5. However, incorporation of glucose increased only 3-folds prior to this conversion, even though GLUT-1 level increased by 13-fold at day 7. GLUT-4, on the other hand, was not detected during the course of differentiation. These results suggested that adipose tissue metabolisms in ruminants were different from that of non-ruminants.

Measles encephalomyelitis in a patient with a history of vaccination.

Secondary vaccine failure (SVF) of measles is generally believed to run a milder course of illness than an ordinary course of infection. Severe complications such as central nervous system involvement have rarely been reported. A 12 year old girl, who had received a live attenuated measles vaccine 10 years earlier, developed an encephalomyelitis in the absence of symptoms indicative of ordinary measles such as Koplik spots. Anti-measles hemagglutination inhibition (HI) titer and measles IgM and IgG antibody titers were measured in a commercial laboratory. Measles virus genomic sequence was detected by polymerase chain reaction. Both serum and cerebrospinal fluid (CSF) samples obtained at acute phase already showed extremely high titers of HI (x8192 in serum and x1024 in CSF, respectively) and IgG antibody along with the presence of IgM antibody. Polymerase chain reaction detected the measles virus genomic sequence in the acute phase CSF. The patient's definite history of measles vaccination, high titers of HI and IgG antibodies observed at the very early stage of illness and the clinical course indicated that this patient has an encephalomyelitis due to SVF of measles. It is suggested that measles virus can be a pathogen of encephalitis without symptoms indicative of ordinary measles in individuals who received live attenuated measles vaccines.

Thermodynamics of formation of urea complexes with manganese(II), nickel(II) and zinc(II) ions in N,N-dimethylformamide.

The complexation of urea (ur) with manganese(II), nickel(II) and zinc(II) ions has been studied by titration calorimetry in N,N-dimethylformamide (DMF) containing 0.4M (C(2)H(5))(4) NBF(4) as a constant ionic medium at 25 degrees C. The calorimetric data were well explained in terms of the formation of [Mn(ur)](2+), [Mn(ur)(2)](2+) and [Mn(ur)(4)](2+) for manganese(II), [Ni(ur)](2+) for nickel(II) and [Zn(ur)](2+) and [Zn(ur)(2)](2+) for zinc(II), and their formation constants, reaction enthalpies and entropies were determined. The complexation of the nickel(II)-urea system in DMF has also been studied by means of spectrophotometric titration and electronic spectra of individual nickel(II) complexes were determined. On the basis of the stepwise thermodynamic quantities and the individual electronic spectra of the complexes, it is revealed that the [Mn(ur)](2+), [Mn(ur)(2)](2+), [Ni(ur)](2+), [Zn(ur)](2+) and [Zn(ur)(2)](2+) complexes have a six-coordinate octahedral structure, while the [Mn(ur)(4)](2+) complex has a four-coordinate tetrahedral structure.

Successful intravenous immunoglobulin therapy for recurrent pneumococcal otitis media in young children.

Serum immunoglobulin levels and naturally occurring antibody titres against Streptococcus pneumoniae were measured in seven children aged 1-1.9 years with recurrent pneumococcal acute otitis media (AOM). Three of them had low IgG2 levels. Mean antibody levels of anti-pneumococcal IgG1 and anti-pneumococcal IgG2 were significantly lower in patients when compared to those of healthy controls and children who had less frequent episodes of AOM. Following treatment with intravenous immunoglobulin (IVIG) for 6 months, anti-pneumococcal IgG1 and IgG2 antibody levels increased and the number of episodes of AOM decreased in all patients. Following the discontinuation of IVIG therapy, no AOM episode occurred. Serum levels of anti-pneumococcal IgG1 and IgG2 were normal, which were measured in three subjects at 5, 6, and 12 months after the cessation of IVIG therapy. These results suggested that delayed maturation of anti-pneumococcal antibody production caused recurrent AOM and this condition was corrected by IVIG therapy.

Structure of iron(III) ion and its complexation with thiocyanate ion in N,N-dimethylformamide.

Complexation of iron(III) with thiocyanate ions has been calorimetrically and spectrophotometrically investigated in N,N-dimethylformamide (DMF) containing 0.4 mol/dm(3) (C(2)H(5))(4)NClO(4) or 1 mol/dm(3) NH(4)ClO(4) as a constant ionic medium at 25 degrees C. Calorimetric titration data were well explained in terms of the formation of [Fe(SCN)(n)]((3-n)+) (n = 1-5), and their formation constants, reaction enthalpies and entropies were determined. Electronic spectra of individual iron(III) thiocyanato complexes were also determined. The stepwise thermodynamic quantities changed monotonously, i.e. DeltaG degrees (1) < DeltaG degrees (2) < DeltaG degrees (3) < DeltaG degrees (4), < DeltaG degrees (5), DeltaH degrees (1) > DeltaH degrees (2) > DeltaH degrees (3) > DeltaH degrees (4) > DeltaH degrees (5), DeltaS degrees (1) > DeltaS degrees (2) > DeltaS degrees (3) > DeltaS degrees (4) > DeltaS degrees (5). This suggests that no extensive desolvation occurred at any step of complexation. On the basis of these thermodynamic quantities, it is postulated that the [Fe(SCN)(n)]((3-n)+) (n = 1-5) complexes have a six-coordinate octahedral structure as well as the [Fe(dmf)(6)](3+) ion, the octahedral structure of which has been confirmed by the EXAFS (extended X-ray absorption fine structure) method.

Establishment of stable mouse/human-human hybrid cell lines producing large amounts of anti-tetanus human monoclonal antibodies with high neutralizing activity.

To establish stable hybrid cell lines producing human anti-tetanus antibody with high toxin-neutralizing activity, peripheral lymphocytes from humans hyperimmunized with tetanus toxoid were, after in vitro antigen stimulation, fused with a mouse/human heteromyeloma or human lymphoblastoid cell line and cloned. Unlike the IgM secretors (six clones), the IgG secretors we obtained (six clones) produced anti-tetanus human monoclonal antibodies with high neutralizing activity (the highest one, cell line G2, 4.3 IU/100 micrograms IgG). Appropriate combinations of three or four kinds of monoclonal antibodies of the IgG type resulted in markedly increased neutralizing activity comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically on the basis of toxin-specific IgG content. Five of these cell lines produced 10-20 micrograms of antibody per ml for more than 3 months. The cell line G2 produced 6 mg of antibody per day in serum-free medium in a 500-ml bioreactor in perfusion culture and 13-104 mg in a nude mouse. These cell lines satisfied, for the first time, the minimal requirements for applying human monoclonal antibodies to clinical use.

Species identification of cooked meats by DNA hybridization assay.

Dot-blots hybridization technique has been applied to the detection of species-specific DNA fragments in the cooked meats of chicken, pig, goat, sheep, and beef. The samples were obtained from the meats which were heated for 30 min at 80, 100 or 120°C. The probes, biotin-labeled chromosomal DNA fragments, were hybridized to the sample DNA on nylon membranes. The species of the meats cooked at 100 or 120°C were identified at 100 ng/dot of the sample DNA. The probes for chicken and pig did not show cross-reactivity, but those for the ruminants reacted with other ruminant DNA. Using this method, chicken, pig and beef were detected from 50 mg of the commercial canned products.

Isolation, purification, and characterization of fragment B, the NH2-terminal half of the heavy chain of tetanus toxin.

Fragment B, the N-terminal half of the heavy chain, an important domain of the tetanus neurotoxin molecule, was isolated for the first time. Tetanus toxin (composed of three domains, A, B, and C) was prepared from culture filtrates. Fragment A-B, derived from the toxin treated mildly with papain, was used for the isolation of fragment B. Fragment A-B obtained was dissociated into fragments A and B by reduction with 100 mM dithiothreitol and treatment with 2 M urea. Fragment B was separated from fragment A by ion-exchange column chromatography on a Mono Q column equilibrated with 20 mM Tris hydrochloride buffer (pH 7.6), containing 1 mM dithiothreitol and 2 M urea, in a fast-protein liquid chromatography system by elution with a linear gradient of 0 to 0.5 M NaCl. Fragment B was obtained in two forms having molecular weights of 48,000 +/- 2,000, which were indistinguishable by sodium dodecyl sulfate-gel electrophoresis or antigenic specificity, but distinguishable on polyacrylamide gel electrophoresis without sodium dodecyl sulfate and on isoelectric focusing (pI 6.7 and 7.3). The recovery of fragment B was 50 to 72% of that of fragment A-B on a molar basis. Purified fragment B was not toxic to mice on intravenous or intramuscular injection at doses of up to 100 micrograms, but was found to form channels (ca. 2.3 pS) in a lipid bilayer membrane by a patch clamp technique. The role of domain B of the tetanus toxin molecule in the mechanism of action of the toxin is discussed.

Isolation and purification by high performance liquid chromatography of a tetanus toxin fragment (fragment [A-B]) derived from mildly papain-treated toxin.

Fragment [A-B] of tetanus toxin was highly purified by combination of gel permeation chromatography, adsorption chromatography in an HPLC and immunoadsorption chromatography using anti-Fragment [C] as a ligand. The purified Fragment [A-B] (200 micrograms) elicited a peculiar toxicity, 'hypoactivity' or 'weakness', and killed the mice in ca. 73 hr and 88 hr when it was injected i.v. and i.m., respectively. However, contamination by the whole toxin was not detectable, in the purified fragment preparation, when up to 600 micrograms was tested by the mouse toxicity assay.

Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane.

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line.

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.

[Thirty-five cases of tuberculosis in childhood].

Thirty five children with tuberculosis admitted to the Hokkaido University Hospital from 1970 through 1984 wer reviewed. They were 7 cases of meningeal tuberculosis, 8 of military tuberculosis, 15 of primary tuberculosis and 5 of adult type pulmonary tuberculosis. Fifteen (43%) of the 35 cases were below 2 years, and over half of these younger cases were hematogenous spread such as meningeal and military types. Instead of hematogenic spread in these cases, erythrocyte sedimentation rate and CRP stayed in moderate values. Thirty one (91.2%) of the 34 cases having precise record had no history of BCG inoculation. Over half of the cases were infected from adults in the immediate household, and the sources of infection were found after the diagnosis of childhood tuberculosis in about half of these cases. Although incidence of the childhood tuberculosis are decreasing in recent years, childhood tuberculosis resulting hematogenous spread with poor prognosis in advanced state still exists, so it is important to remark the childhood tuberculosis at out patient clinics.

Rapid method for purification of Clostridium botulinum type C neurotoxin by high performance liquid chromatography (HPLC).

The culture supernatant of Clostridium botulinum type C, concentrated by addition of RNA, acid precipitation and subsequent protamine treatment was used as starting material for rapid purification of L toxin (mol. wt. ca. 500K) and M toxin (mol. wt. ca. 350K) of C1 neurotoxin by ion-exchange chromatography on a Mono S column by fast performance liquid chromatography (FPLC). L and M toxins were highly purified further by gel permeation chromatography through a TSK G3000SW column at pH 6.0 by high performance liquid chromatography (HPLC). Purified S toxin (mol. wt. ca. 150K, C1 neurotoxin without a nontoxic component) was then obtained from L toxin rapidly by gel permeation chromatography at pH 7.3 through a TSK G3000SW column by HPLC. Purified S toxin was also obtained rapidly from M and L toxins by ion-exchange chromatography on a Mono Q column at pH 8.0 using an FPLC system. The purified preparations of L, M and S toxins gave single bands on conventional polyacrylamide gel electrophoresis, and had specific activities of 2.8, 6.7, and 14-21 X 10(7) LD50/mg N, respectively, in mice. On immunoelectrophoresis, purified S toxin gave a single arc against anti-crude toxin serum. The yield of toxicity as L and M toxins was 73.1% (32.5% as L toxin and 40.6% as M toxin) from the protamine-treated concentrated culture supernatant. The recovery of toxicity as S toxin from purified L or M toxin was almost 100% (97.6-100% of L toxin and 97.5% of M toxin). These procedures provide a rapid method for purifying L and M toxins, which have stable toxicities.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary action of Clostridium perfringens type A enterotoxin on HeLa and Vero cells in the absence of extracellular calcium: rapid and characteristic changes in membrane permeability.

Clostridium perfringens type A enterotoxin bound rapidly to HeLa and Vero cells in the absence of extracellular Ca2+ at 37 degrees C. The bound toxin rapidly (within 2 min) caused influx of Na+ and efflux of K+ and Mg2+. Changes in membrane permeability occurred in the absence or presence of extracellular Ca2+ and to the similar extents at 37 degrees C and 4 degrees C, in contrast to the subsequent bleb and balloon formation, which required both extracellular Ca2+ and incubation at 37 degrees C. Substances with molecular weights of over ca. 200 protected the cells from the morphological alterations induced by the toxin, whereas substances with molecular weights of less than ca. 200 did not. The mechanism of the primary action of the enterotoxin is discussed.

Rapid method for purification of Clostridium botulinuh type C neurotoxin by High Performance Liquid Chromatography (HPLC ).

The culture supernatant of Clostridium botulinum type C, concentrated by addition of RNA, acid precipitation and subsequent protamine treatment was used as starting material for rapid purification of L toxin (mol. wt. ca. 500K) and M toxin (mol. wt. ca. 350K) of C1 neurotoxin by ion-exchange chromatography on a Mono S column by fast performance liquid chromatography (FPLC). L and M toxins were highly purified further by gel permeation chromatography through a TSK G3000SW column at pH 6.0 by high performance liquid chromatography (HPLC). Purified S toxin (mol. wt. ca. 150K, Cl neurotoxin without a nontoxic component) was then obtained from L toxin rapidly by gel permeation chromatography at pH 7.3 through a TSK G3000SW column by HPLC. Purified S toxin was also obtained rapidly from M and L toxins by ion-exchange chromatography on a Mono Q column at pH 8.0 using an FPLC system. The purified preparations of L, M and S toxins gave single bands on conventional polyacrylamide gel electrophoresis, and had specific activities of 2.8, 6.7, and 14-21 × 107 LD50/mg N, respectively, in mice. On immunoelectrophoresis, purified S toxin gave a single arc against anti-crude toxin serum. The yield of toxicity as L and M toxins was 73.1% (32.5% as L toxin and 40.6% as M toxin) from the protamine-treated concentrated culture supernatant. The recovery of toxicity as S toxin from purified L or M toxin was almost 100% (97.6-100% of L toxin and 97.5% of M toxin). These procedures provide a rapid method for purifying L and M toxins, which have stable toxicities. The method will also be very useful for rapid preparation of the toxic component (S toxin) of C1 neurotoxin, which is unstable, in small amounts from the L and M toxins just before its use in experiments.

Morphological alterations and changes in cellular cations induced by Clostridium perfringens type A enterotoxin in tissue culture cells.

The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions. The action of enterotoxin was found to depend on the temperature but not on energy metabolism. The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca2+ and Mg2+. Of the constituents of the buffered saline, Ca2+ was essential for the morphological alterations and other ions were interchangeable. The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca. 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl2 at a concentration of over ca. 50 mM. Addition of sucrose to the medium prevented induction of the morphological alterations. The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl2 concentration in the medium. A calcium ionophore A23187 mimicked the action of enterotoxin. Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca2+ during intoxication with concomitant changes in Na+, K+ and Mg2+ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment. The mechanism of action of C. perfringens type A enterotoxin is discussed on the basis of these findings.