Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.     

Particle size of airborne mouse crude and defined allergens

Laboratory animal allergy is a serious occupational diseases of many workers and scientists engaged in animal experimentation. Control measures depend upon characterization of allergens including airborne particles. This study measured the particle size of crude mouse urine and pelt aeroallergens generated in mouse housing rooms and compared them with mouse serum albumin, a defined major allergen. Allergens were detected by specific immunological methods. Most crude and defined allergens (74.5-86.4%) concentrated on a filter with a retention size greater than 7 microns. In distrubed air, allergen concentration increased 1.4 (albumin) to 5 (crude) fold and the proportion of small particles increased from 1.4% in calm air to 4.5% in distrubed air. This information on the generation and size distribution of aeroallergens will be important in the development of effective counter measures.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Functional analysis of the γ-glutamylcysteine synthetase of Escherichia coli B: effect of substitution of His-150 to Ala

A high expression system of the -glutamylcysteine synthetase gene (gshl) of Escherichia coli B was constructed, and rapid purification of GSH-I was performed. The active site of GSH-I was analysed by chemical modification, and Lys, Arg and His residues seemed to be involved in the active site of the enzyme. Among them, His residues were substituted to Ala by site-directed mutagenesis, and His-150 was found to be essential for the activity of GSH-I. Correspondence to: A. Kimura     

A new method of inhalation anesthesia with nasopharyngeal insufflation in rat experiment.

We have established a new method of anesthesia with nasopharyngeal insufflation for intraoral procedure in rodents. Twelve male Wistar rats weighing 330-390 g were used in this study. Insertion of a feeding tube 1.0 mm in diameter coated with 2% xylocaine jelly was inserted into the nasal cavity approximately 25 mm from the naris, and anesthetization with mixed gas of 100% oxygen with 3-4% enflurane at 0.25-0.5 l/min flow rate was achieved. Using this anesthetic method, a chronic experiment comprising 1-h/day experimental procedure was carried out for 14 days. This method enabled, 1) simple and safe operation of the induction, emergence and anesthetic depth, 2) experimental procedures on the dental/oral region, 3) avoidance of the dyspnea and tachypnea, and 4) avoidance of cumulative effects in daily anesthesia.     

High-frequency vibration in flagellar axonemes with amplitudes reflecting the size of tubulin

Flagellar axonemes of sea urchin sperm display high-frequency (approximately 300 Hz) vibration with nanometer-scale amplitudes in the presence of ATP (Kamimura, S., and R. Kamiya. 1989. Nature (Lond.). 340:476-478). The vibration appears to represent normal mechanochemical interaction between dynein and microtubules because the dependence of the frequency on MgATP concentration is similar to that of the axonemal motility, and because it is inhibited by micromolar concentrations of vanadate. In this study a two-dimensional photo-sensor was used to characterize this phenomenon in detail. Several new features were revealed. First, the vibration was found to be due to a back-and-forth movement of the doublet microtubules along the axonemal length. Two beads attached to different parts of the same axoneme vibrated in unison, i.e., synchronized exactly in phase. This suggested that the outer doublet can be regarded as a stiff rod in vibrating axonemes. Second, evidence was obtained that the amplitude of the vibration reflected the number of active dynein arms. Third, under certain conditions, the vibration amplitude took stepwise values of 8 x N + 4 nm (N = 0, 1, 2, 3, or 4), indicating that the amplitude of microtubule sliding was limited by the size of tubulin dimer (8 nm) or monomer (4 nm). To explain this phenomenon, a model is presented based on an assumption that the force production by dynein is turned off when dynein is subjected to tensile force; i.e., dynein is assumed to be equipped with a feedback mechanism necessary for oscillation.     

Tubulin protofilaments and kinesin-dependent motility

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.