Self-binding antibodies (autobodies) form specific complexes in solution

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese,Japanese and South Korean families reveals dominance of the mutant allele

Summary Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.     

Cytochemical properties of osteoblast cell membrane domains

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.     

Characterization of Senescence in Lemna gibba G3: A Determinate Growth System

Frond senescence in Lemna gibba G3 was characterized, and itscontrol by light, ABA and kinetin investigated. The plant exhibitsa determinate growth pattern with a frond producing a set numberof daughter fronds before undergoing senescence and death regardlessof whether or not it flowers. When a frond was cut in half,the distal half (half frond) which lacks any meristem underwentrapid senescence as compared with intact fronds. In both intactand half fronds, the onset of senescence was accelerated byABA and retarded by kinetin. Continuous white light acceleratedsenescence in both intact and half fronds over the dark controls.Under different photoperiodic light regime, the pace of daughterfrond production is accelerated in proportion to the lengthof light period. In half fronds, however, very short photoperiodiclight treatments (e.g. 1L: 23D or 3L: 21D) rather delayed senescenceover the dark controls. Two separate light control systems operatingin opposite directions in Lemana senescence appear to exist. ¹Present address: Department of Biology, Yonsei University,Seoul 120-749, Korea ²Present address: U.S. Department of Agriculture, Aero SpaceBuilding, Rm. 323, 901 D Street, S.W., Washington, D.C. 20251-2200, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.     

Characterization of "depolarization"-induced calcium release from sarcoplasmic reticulum in vitro with the use of membrane potential probe.

In the triad, the complex of transverse (T) tubule and sarcoplasmic reticulum (SR) Ca2+ release is induced from SR by mediation of the T-tubule. We report here evidence that this Ca2+ release is produced by depolarization of the T-tubule moiety. Thus, we found that the amount of [14C]SCN- taken up by T-tubules and triads (but not that by SR) increased upon incubation with (K, Na) gluconate, Mg ATP, indicating that the T-tubule was polarized making the lumenal side (equivalent to the extracellular side of an intact muscle fiber) more positive. Upon mixing with choline chloride, the procedure to induce Ca2+ release, [14C]SCN- uptake decreased, indicating that the T-tubule became depolarized. Activation of the T-tubule polarization by Na+ and prevention of it by digoxin [inhibitor of the (Na+, K+) pump], respectively, led to activation and inhibition of choline chloride-induced SR Ca2+ release.     

Kinetics of producing pro-urokinase in perfusion cultivations

Summary Better production of pro-urokinase from human cell line was observed with 5% serum containing medium than 10% or serum free medium on Cytodex II under perfusion chemostat operations, showing 0.8×10^–5 (IU/daycell) of maximum productivity at 0.020 (l/h) of dilution rate in 5% serum medium, which corresponds to 800 IU/mL at this dilution rate. Conversion of pro-urokinase was reduced in the serum-containing media.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

神经敏感性的降低是棉铃虫对拟除虫菊酯抗药性的重要机制

用扫描电子显微镜和光学显微镜的染色观察,研究了棉铃虫Helicoverpa armigeraHubner 3龄幼虫腹部神经一肌肉的分布,表明其腹纵肌细胞适合于作电生理细胞内记录。用电生理细胞内微电极记录法研究了棉铃虫三个品系：对照品系、功夫菊酯抗性品系(Cy-R,抗性指数13.4)和氰戊菊酯抗性品系(Fn-R,抗性指数37.9)的3龄幼虫神经一肌肉材料对相应杀虫剂的神经敏感性。结果表明：用10^-6mol/L的功夫菊酯处理Cy-R,在30min内有40%的个体对药剂无反应,另外60%的个体产生反应所需的时间是对照品系的3.5倍。用10^-6mol/L的氰戊菊酯处理Fn-R,在30min内有47%的个体对药剂无反应,另外53%的个体产生反应所需的时间是对照品系的4.2倍。这些结果说明神经敏感性降低是棉铃虫对拟除虫菊酯抗药性的重要机制。