Monoclonal antibodies to human brain acetylcholinesterase: properties and applications

1. Acetylcholinesterase (AChE) was purified 20,000-fold in a 43% yield from 90 g of human cerebellum by combined immunoaffinity and ligand affinity chromatography. The purified enzyme migrated as a 68,000-dalton band during polyacrylamide gel electrophoresis under denaturing and reducing conditions. 2. Balb/c mice were immunized with multiple 10-micrograms injections of this material in order to raise monoclonal antibodies to human brain AChE. Three such antibodies were obtained and characterized. 3. Each antibody cross-reacted distinctively with AChEs from other mammals. No antibody recognized human plasma butyrylcholinesterase but all reacted with AChE from human red blood cells. 4. Antibodies HR5 and HR3 performed well in two-site immunoassays for AChE. With these assays we compared autopsy samples of cortical region A9 from six controls (nonneurological cases) and five patients with Alzheimer's disease. The latter showed a highly significant 60% deficit of AChE protein. 5. The present antibodies will permit additional immunochemical studies of cholinergic systems in dementia.     

Three regulatory nodD alleles of diverged flavonoid-specificity are involved in host-dependent nodulation by Rhizobium meliloti

Summary Rhizobium meliloti infective on Medicago, Melilotus and Trigonella plants has three copies of the nodulation regulatory gene nodD. Strains containing mutations in nodD1 exhibited a delayed and/or decreased nodulation on Melilotus albus (Ma), Medicago sativa (Ms), Medicago quasifalcata (Mqu) and Trigonella coerulea (Tc), while on Medicago truncatula (Mt) they nodulated similarly to the wild-type R. meliloti. Delayed nodulation was observed also when nodD2 mutants were inoculated onto Ms, Mt and Tc, but not on Ma and Mqu. A nodD3 mutant exhibited delayed nodulation on Ms and Ma. Using a nodC-lacZ fusion and cloned nodD genes on plasmids, high induction levels were detected in R. meliloti when nodD1 was present with seed exudates from Ms, Ma and Mqu, nodD2 with those from Ms and Mt, and nodD3 with those from Ms, Ma and Mqu. NOne of the nodD copies exhibited high levels of nodC-lacZ induction when present with seed exudate from Tc. Only nodD1 induced nodC-lacZ expression in conjunction with the flavone, luteolin. The plant hosts used in this study exude different flavonoids and correlation between nodulation and nodC-lacZ induction abilities of the host exudates was observed. We concluded that all the three nodD copies of R. meliloti have common nod-promoter activating but diverged flavonoid-recognizing abilities. Thus, the three nodD alleles contribute to the activation of nodulation genes in a host-dependent manner.     

Cold-tolerant (psychrotrophic) moulds and blue stain fungi from softwood in Sweden. Growth rates in relation to pH and temperature

Seventy isolates of moulds and blue stain fungi ( Zygomycota and Deuteromycota ), isolated from discoloured outdoor softwood in Sweden, comprising of 27 different species, (the two largest genera Penicillium and Cladosporium ) were investigated for their linear growth at three different start-pH values (5, 7 and 9.5) at two temperatures (2°C and 24°C) on malt extract agar (MEA). At 24°C all isolates showed growth at all three start-pH values except for one isolate which did not grow at initial-pH 9.5. After 21 days at 2°C at the three start pH-values, only six isolates showed no growth indicating that 64 of the isolates were cold-tolerant (psychrotrophic). Of these 64 strains, 58 showed growth at an initial pH of 9.5. Lower pH optima at 2°C than at 24°C were found for most of the isolates. The reduction of the linear growth at initial pH 9.5 in relation to the growth at optimal pH was more pronounced (higher) at the low temperature.     

Development of sertoli cell junctions in vitro—A freeze-fracture study

Summary Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo. This study was supported by Grant 801D185 (Dr. Posalaky) from the Medical Education and Research Foundation, St. Paul-Ramsey Hospital.     

Receptor specificity,functional characteristics,and cell-surface phenotype of a highly selected anti-I-Ab-specific,long-term T-cell line

A highly selected alloreactive T-cell line was developed by repeated restimulation of B10.D2/n lymph-node cells with irradiated C57BL/10Sn (BIO) spleen cells in long-term MLC for up to 2 1/2 years. Continuous growth of the line requires restimulation every 2 to 4 weeks with fresh H-2^b stimulator cells. The line proliferates strongly against H-2^b but not againstH-2 ^d ,H-2 ^f ,H-2 ^q ,H-2 ^r , orH-2 ^s stimulators. Analysis of recombinant mouse strains showed that the proliferative response is directed against I-A^b but not K^b or D^b determinants. During the growth period of the line, strong cross-reactivity with H-2^p (B10.P) and weak cross-reactivity with H-2^k strains (e.g., CBA/J and B10.BR) was observed. A clone with exquisite specificity for I-A^b, but with no cross-reactivity with H-2^p or H-2^k was isolated from the line; thus clonal heterogeneity of the line still exists despite the highly selective growth conditions. — The majority of T cells from the line or clone were shown to bind I-A^b but not K^b or D^b determinants either spontaneously during restimulation with fresh B10 stimulator cells or via membrane vesicles expressing I-A^b determinants. No killing activity by the line in either specific or nonspecific cytolytic T-cell assays was observed nor was the T 145 glycoprotein, characteristic of killer T cells, detected.Abbreviations used in this paper B6 C57BL/6J - B10 C57BL/10Sn - Con A Concanavalin A - CTL cytotoxic T lymphocyte - FCS fetal calf serum - FDA fluorescein diacetate - FITC fluorescein isothiocyanate - Ia I-region-associated antigens - LPS lipopolysaccharide fromE. coli - Lyt T-lymphocyte-defined antigen - MLC mixed leukocyte culture - NP-40 nonidet P-40 - PAGE pofyacrylamide gel electrophoresis - PHA phytohemagglutinin fromPhaseolus vulgaris - PM plasma membrane - SDS sodium dodecyl sulfate - TCGF T-cell growth factor(s) - TdR thymidine     

Involvement of Mhc Loci in immune responses that are not Ir-gene-controlled

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymphnode cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative response of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time -antibodies specific for the A (AA) or E (EE) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastoinyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Irr genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of Ir-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by Ir genes: both types involve the recognition of the antigen, in the context of Mhc molecules.Abbreviations used in this paper A class II MHC molecule consisting of the A and A chains - ADH alcohol dehydrogenase - APC antigen-presenting cell - cpm counts per minute - E class II Mhc molecule onsisting of the E and A chains - Ir immune response - KLH key-hole limpet hemocyanin - Mhc major histocompatibility complex - PBS phosphate buffered salt solution - SD standard deviation - SI stimulation index - SN supernatant of Sendai-virus preparation     

Frequency-dependent selection on information-transfer strategies at breeding colonies: a simulation study

Through computer simulations, we model three different foodfinding strategies: searcher, no information transfer, watcher,limited information transfer; follower, full information transfer.The aim of this article was to study how frequency-dependentselection affects the proportion of these strategies at a simulatedcolony under different patterns of food distribution. Furthermore,we determined how information transfer in a population witha mixed evolutionarily stable strategy (ESS) modified the averageforaging efficiency of an individual compared to that of anindividual in a population with mutual information exchange.We found that the proportion of information gaining strategiesincreases as the food resources become more clumped. The improvementin foraging efficiency through the operation of an informationcenter need not require mutuality in information exchange. Onthe basis of the presented study, at the ESS only a small percentageof colony members need discover food patches, yet the foragingefficiency may be high because of the operation of an informationcenter.     

Reconstitution of chlorophylls and photosynthetic CO2 assimilation upon rehydration of the desiccated poikilochlorophyllous plant Xerophyta scabrida (Pax) Th. Dur. et Schinz

Resynthesis of the photosynthetic apparatus and resumption of CO₂ assimilation upon rehydration is reported for the monocotyledonous and poikilochlorophyllous desiccation-tolerant (PDT) plant Xerophyta scabrida (Pax) Th. Dur. et Schinz (Velloziaceae). During desiccation there was a complete breakdown of chlorophylls whereas the total carotenoid content of air-dried leaves was reduced to about 22% of that of functional leaves. The prerequisites for the resynthesis of photosynthetic pigments and functional thylakoids were the reappearance of turgor and maximum leaf water content at 2 and 10 h after rehydration, respectively. The period of increased initial respiration after rewetting leaves (rehydration respiration) lasted up to 30 h and was thus 6 to 10 times longer than in homoiochlorophyllous desiccation-tolerant plants (HDTs) in which chlorophylls are retained during desiccation. Accumulation of chlorophylls a + b and total carotenoids (xanthophylls and carotene) started 10 h after rehydration. Normal levels of chlorophyll and carotenoids were obtained 72 h after rehydration. Values for the variable-fluorescence decrease ratio (Rfd690 values), an indicator of photochemical activity, showed that photochemical function started 10 h after rehydration, but normal values of 2.7 were reached only 72 h after rehydration. Net CO₂ assimilation started 24 h after rewetting and normal rates were reached after 72 h, at the same time as normal values of stomatal conductance were obtained. The increasing rates of net CO₂ assimilation were paralleled by decreasing values of the intercellular CO₂ concentration. All photosynthetic parameters investigated showed values normal for functional chloroplasts by 72 h after the onset of rehydration. Fully regreened leaves of the presumed C₃ plant X. scabrida exhibited a net CO₂ assimilation rate which was in the same range as that of other C₃ plants and higher than that of recovered HDT plants. The fundamental difference between air-dried PDT plants, such as X. scabrida, which have to resynthesize the photosynthetic pigment apparatus, and air-dried HDT plants, which only undergo a functional recovery, is discussed.Abbreviations c -carotene - c_i intercellular CO₂ concentration - Car x + c total carotenoid content x + c - Chl a + b total chlorophyll a + b content - g_s stomatal conductance - HDT homoiochlorophyllous desiccation tolerant - LWC leaf-water content - P_N net photosynthesis rate - PDT poikilochloro phyllous desiccation tolerant - R_d dark respiration - Rfd variable fluorescence decrease ratio (Rfd = fd/fs) - x xanthophylls The senior author thanks the Deutschem Akademischem Auslandsdienst (Bonn, Germany), Soros Foundation (Budapest, Hungary) and European Community (Brussels, Belgium) for providing fellowships for research periods at Karlsruhe. The research was also supported by the Hungarian Scientific Research Foundation (OTKA I/848, OTKA I/3.1545 and OTKA I/4.F.5359). We wish to thank Professor T. Pocs (Eger, Hungary — Morogoro, Tanzania) for collecting the plant material and to the linguist Mr. A. Jackson for correcting the English.     

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.     

Homology of the ligand-binding regions of Rhizobium symbiotic regulatory protein NodD and vertebrate nuclear receptors.

Summary The signal specificity and structure of sensor-activator proteins from different species (NodD of Rhizobium bacteria and vertebrate nuclear receptors) were compared. Several compounds (including flavonoids, coumestrol and estradiol) that bind to mammalian receptors also interact with NodD proteins. NodD-dependent synergism of the signal compounds luteolin and catechin was observed suggesting that these compounds bind directly to NodD. Two regions comprising 63 and 37 amino acids in NodD showed 45% and 36% homology, respectively, with the estrogen receptor. These regions, designated as modules M1 and M2, coincide with conserved parts of the ligand-binding domains of the nuclear receptors. A part of NodD overlapping with the M1 module was predicted to be membrane associated and was 46% homologous to a membrane-spanning sensory segment of the Agrobacterium VirA protein. We suggest that the homologous polypeptide modules detected in NodD and the nuclear receptors originate from a common ancestor protein and may be directly involved in ligand binding.