Energy transfer distance measurements in immunoglobulins. II. Localization of the hapten binding sites and the interheavy chain disulfide bond in rabbit antibody

The distance between the hapten combining site and the single interheavy chain disulfide bond in rabbit immunoglobulin G has been determined by measuring the efficiency of energy transfer between chromophores specifically attached at these sites on the molecule. The donor chromophore, DnsLys³, was non-covalently bound in the combining sites of high-affinity antiDns antibody molecules, in one case, and in the combining site of the pepsin Fab′ fragment of antiDns in another. The acceptor chromophore, fluorescein, was covalently attached by disulfide interchange of di-FlCys with sulfhydryls generated by selective reduction of the interheavy chain disulfide bond of whole antiDns antibody and of the (Fab′)₂ pepsin fragment. The presence of acceptor decreased the donor fluorescence lifetime by about 1.0 nanosecond in both cases, i.e. for the whole antibody, from 23.6 to 22.7 nanoseconds, and for the Fab′ fragment from 23.6 to 22.5 nanoseconds. An average separation distance of 81 Å was calculated from an average observed transfer efficiency of 3.7%. This value agrees closely with the over-all length of a Fab′ fragment of a human IgG myeloma protein (Poljak et al., 1972). These results strongly suggest that the antibody combining site is at, or very close to, the tip of the Fab fragment and that the inter-heavy chain disulfide bond is at or near the edge of the C_L?C_H1 domain.     

Wirkungen von Rohöl-Emulgatorgemischen auf marine Fischbrut und deren Nährtiere

Zusammenfassung 1. Es wurde die Einwirkung eines Rohöl-Emulgatorgemisches (Irak-Öl/Moltoclar) auf die Larven vonClupea harengus L. undAgonus cataphractus L. sowie auf Wildplankton untersucht.2. Bei Emulgatorkonzentrationen von 2,5 bis 5,0 mg/l war die Letalitätsgrenze erreicht.3. Subletale Schädigungen ließen sich bis zu einer Konzentration von 0,5 mg/l deutlich nachweisen.4. Irak-Rohöl hatte keine schädigenden Wirkungen auf Heringslarven.

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Effects of crude oil-emulsifier mixtures on marine fish fry and their food animals

The effects of crude oil-emulsifier mixtures on the larvae ofClupea harengus L. andAgonus cataphractus L. are described. The herring larvae tested had total lengths of 20 to 26 mm; the larvae ofAgonus cataphractus were investigated shortly after their first food intake. Samples of wild plankton from daily catches (used as food during rearing experiments) were also tested. The different dilutions were obtained from the following initial mixture: 20 ml emulsifier Moltoclar, 80 ml Irak crude oil, 900 ml stale sea water (32 S ). Dilutions with the following content of the emulsifier were used: 50 mg/l, 25 mg/l, 5 mg/l, 2.5 mg/l, 0.5 mg/l, 50µg/l, 5µg/l, 0.5µg/l. Lethal concentrations ranged from 2.5 to 5.0 mg/l. Sublethal damages were clearly ascertained down to a concentration of 0.5 mg/l. Irak crude oil alone did not cause damage to herring larvae during the observation period of 4 days.

     

Ovariolenverlängerung und eiablage bei der termite Kalotermes flavicollis Fabr

Zusammenfassung Die Oogenese der Termite Kalotermes flavicollis Fabr. wurde nach der Form der Oocyten in Phasen gegliedert (Abb. 2). Die panoistisehen Ovariolen enthalten in linearer Anordnung Oocyten der verschiedensten Entwicklungsstadien. Beide Ovarien haben insgesamt 14 Ovariolen (Abb. 1). Diese Zahl findet sich in Larven, Nymphen, Soldaten und in Geschlechtstieren unterschiedlichen Alters. Die einzelnen Ovariolen eines Weibchens enthalten unterschiedliche Anzahlen von Oocyten. Es reifen immer nur einige Oocyten gleichzeitig (Abb. 6, 7).Junge Geschlechtstiere legen anfangs nur wenige Eier. Die Eiproduktion erhöht sich später mit dem Ansteigen der Zahl der Individuen in der Kolonie. Eine erhebliche Erhöhung der Eiproduktion ist bei alten Geschlechtstieren aus großen Völkern festzustellen. Diese Erhöhung der Eiproduktion kommt dadurch zustande, daß die Ovariolen in die Länge wachsen und sich die Zahl der Oocyten im Vitellarium erhöht (Abb. 5a, b, c und 8a, b, c). Es besteht nämlich eine Korrelation zwischen der Ovariolenlänge und der Zahl der abgelegten Eier pro Zeiteinheit. Die Verlängerung der Ovariolen kommt unter dem Einfluß des Volkes zustande. Es kommt zu keiner Verlängerung der Ovariolen bei Weibchen, die länger als zwei Jahre nach dem Schwärmen isoliert gehalten werden. Die Verlängerung der Ovariolen könnte durch die Menge und Art der Nahrung oder ouch durch Wirkstoffe zustande kommen.Der Dottereinbau setzt erst ein nach der Geschlechtstierhäutung, die zu einem imaginalen oder neotenischen Geschlechtstier (Ersatzgeschlechtstier) führen kann. Der Zeitpunkt des Einsetzens des Dottereinbaus fällt bei der Imago zusammen mit dem Einsetzen der Pigmentierung. Es besteht eine Korrelation zwischen Zahl der dottereinbauenden Oocyten pro Ovariole und der zunehmenden Verlängerung der Ovariolen. Es wurden his zu sieben dottereinbauende Oocyten in einer Ovariole festgestellt (Abb. 8a, b, c).

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Summary A subdivision of the oogenesis in Kalotermes flavicollis Fabr. (Isoptera) is described according to the form of the oocytes (Fig. 2). Each ovariole (panoistic type) contains a linear array of oocytes in varying stages of development. Both ovaries together consist of 14 ovarioles (Fig. 1). This number is found in larvae, nymphs; soldiers; in young reproductives and in reproductives aged some years. The number of oocytes varies in the ovarioles of a female. Mature eggs occurs at the same time only in few of ovarioles (Fig. 6, 7).Young reproductives lay only few eggs. The production of eggs increases with increasing number of individuals of the colony. A marked increase of egg production is observed in old reproductives of big colonies. This increase of egg production results from growth in length of the ovarioles and from an increase of the number of oocytes in the vitellarium (Fig. 5a, b, c and 8a, b, c). The length of the ovarioles is correlated with the number of eggs laid within a certain period. The lengthening of the ovarioles is caused by influences from the individuals of the colony. No lengthening of ovarioles took place in females kept singly for more than two years after swarming. The lengthening of the overioles seems to depend on the quantity and the quality of food and/or vitamins.The deposition of yolk takes place only after moulting into a reproductive (imago or neoteinic = replacement reproductive). The start of yolk deposition occurs simultaneously with the start of pigmentation. The number of yolk depositing oocytes increases with the increase of the length of the ovariole. Up to seven yolk depositing oocytes were observed in each single ovariole (Fig. 8a, b, c).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

pH-Dependent association-dissociation of high and low activity plasma a-l-fucosidase

Summary Population and family studies have confirmed the existence of a plasma a-l-fucosidase polymorphism in humans and the autosomal recessive inheritance of the low activity trait. The frequency of the latter is estimated at 11%. The low activity individuals or variants can also be distinguished by the fact that their plasma a-l-fucosidase is heat-inactivated at acidic pH. Sucrose gradient centrifugation results indicate the transition of non-variant plasma a-l-fucosidase with a molecular weight of 66,000 at pH 8.4 to an enzyme form with a molecular weight of 193,000 at pH 3.0. The former is thermolabile, the latter thermostable. Interconversion is pH-dependent. It is hypothesized that the non-variant enzyme, a monomer at alkaline pH, changes upon acidification into a trimeric conformation via dimerization. The thermolabile variant a-l-fucosidase monomer is not converted into a trimer, but only partially into a dimer.     

Aechmea itapoana,a new species from Bahia (Brazil): Taxonomy and ecology

Aechmea itapoana is a new, conspicuous and distinct species from the coastal sand dune scrub of N.E. Brazil. Its possible relationships within subgenusChevaliera, toA. multiflora andA. rodriguesia are discussed.     

Genetics of the apolipoprotein E-system in man

The polymorphism of apolipoprotein E (Apo E) in man is controlled by two codominant alleles, Apo Eⁿ and Apo E^d, at the Apo E-N/D locus and by two alleles, the dominant, Apo E4⁺, and the recessive, Apo E4^o, at the Apo E4 locus.

Frequency distribution analysis of Apo E phenotypes demonstrated a highly significant association between both systems (P ~ 1%). The Apo E4-(+) variant was about twice as frequent in phenotype Apo E-N (30.1%) than in phenotype Apo E-ND (16.4%). The phenotypic combination Apo E-D/-E4(+) was not observed. The segregation of Apo E phenotypes in informative matings is consistent with a close linkage of both loci.

The results may be explained by different models. On the basis of the present data, these models cannot be distinguished by formal genetic criteria. (1) Haplotypes Apo Eⁿ/E4⁺, Apo Eⁿ/E4^o, and Apo E^d/E4^o determine the different phenotypes, and a linkage disequilibrium exists of Δ = .0147 between the E-N/D and E4 loci. (2) The fourth haplotype, Apo E^d/E4⁺, exists, but the gene E4⁺ is not expressed in coupling with Apo E^d. The four-haplotype model seems more attractive in view of Apo E-N/D polymorphism's quantitative character and of biochemical results, which show that phenotypes Apo E-N and Apo E-D differ in the apparent molecular weight (M_r) of the respective major Apo E polymorphic form. Hence, the Apo E-N/D locus may control structural genes involved in the posttranslational modification of Apo E. (3) Finally, there may exist only one Apo E structural gene locus but with mutations at two sites susceptible to posttranslational modification.

     

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: Comparison of proliferating and resting growth states and cell cycle-dependent activity changes

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)