Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG

The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.     

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A+C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial beta' and chloroplast beta' and beta" subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial beta' lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.     

Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain

The first long tract to form in the brain of a vertebrate embryo is the ventral longitudinal pathway. In order to investigate what chemical cues may guide nerve growth cones along this pathway, affinity-purified antibodies to laminin and collagen type IV were used to stain sections of mouse embryos from Embryonic Days 8 through 17. A monoclonal anti-neurofilament antibody was used to show the development of the ventral longitudinal pathway in relationship to immunoreactivity for laminin and collagen type IV. At Day 8 fluorescent immunoreactivity for laminin is bright in the external limiting membrane of the neural tube, but the neuroepithelium does not show bright laminin or neurofilament immunoreactivity. At E9 the ventral longitudinal pathway is forming and punctate immunoreactivity for laminin is present on the surfaces of neuroepithelial cells in the marginal zone, through which axons of the ventral pathway extend. Punctate immunofluorescence for laminin remains concentrated in the marginal zone on Days E10 through E14, but on E16 punctate immunofluorescence was much reduced, although immunoreactivity for laminin remained bright in the maturing pial and arachnoid membranes and on blood vessels in the brain. Immunoreactivity for collagen type IV was strong in the external limiting membrane and on blood vessels, but never showed concentrated punctate immunofluorescence in the marginal zone. These results indicate that laminin may be available on cell surfaces and in extracellular spaces as an adhesive ligand for growth cones during the formation of the ventral longitudinal pathway.     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.     

Spatiotemporal receptive fields: a dynamical model derived from cortical architectonics

We assume that the mammalian neocortex is built up out of some six layers which differ in their morphology and their external connections. Intrinsic connectivity is largely excitatory, leading to a considerable amount of positive feedback. The majority of cortical neurons can be divided into two main classes: the pyramidal cells, which are said to be excitatory, and local cells (most notably the non-spiny stellate cells), which are said to be inhibitory. The form of the dendritic and axonal arborizations of both groups is discussed in detail. This results in a simplified model of the cortex as a stack of six layers with mutual connections determined by the principles of fibre anatomy. This stack can be treated as a multi-input-multi-output system by means of the linear systems theory of homogeneous layers. The detailed equations for the simulation are derived in the Appendix. The results of the simulations show that the temporal and spatial behaviour of an excitation distribution cannot be treated separately. Further, they indicate specific processing in the different layers and some independence from details of wiring. Finally, the simulation results are applied to the theory of visual receptive fields. This yields some insight into the mechanisms possibly underlying hypercomplexity, putative nonlinearities, lateral inhibition, oscillating cell responses, and velocity-dependent tuning curves.     

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12

Summary Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1^intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1^intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1^intB indicated that this element was integrated at the same attP site as previously identified for SSV1^intA. A comparison of the DNA sequences at the left attachment sites of SSV1^intA and SSV1^intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 by inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed. Offprint requests to: W. Zillig     

Misting and nitrogen fertilization of shoots of a saltmarsh grass: effects upon fungal decay of leaf blades

We conducted a 12-week field manipulation experiment in which we raised the nitrogen availability (ammonium sulfate fertilization to roots) and/or water potential (freshwater misting) of decaying leaf blades of a saltmarsh grass (smooth cordgrass, Spartina alterniflora) in triplicate 11-m² plots, and compared the manipulated plots to unmanipulated, control plots. The ascomycetous fungi that dominate cordgrass leaf decomposition processes under natural conditions exhibited a boosting (>2-fold) of living standing crop (ergosterol content) by misting at the 1 st week after tagging of senescent leaves, but afterwards, living-fungal standing crop on misted blades was equivalent to that on control blades, confirming prior evidence that Spartina fungi are well adapted to natural, irregular wetting. Misting also caused 2-fold sharper temporal declines than control in instantaneous rates of fungal production (ergosterol synthesis), 5-fold declines in density of sexual reproductive structures that were not shown by controls, and 2-fold higher rates of loss of plant organic mass. Extra nitrogen gave a long-term boost to living-fungal standing crop (about 2-fold at 12 weeks), which was also reflected in rates of fungal production at 4 weeks, suggesting that saltmarsh fungal production is nitrogen-limited. Although bacterial and green-microalgal crops were boosted by manipulations of nitrogen and/or water, their maximal crops remained 0.3 or 2% (bacteria or green microalgae, respectively) of contemporaneous living-fungal crop. The fungal carbon-productivity values obtained, in conjunction with rates of loss of plant carbon, hinted that fungal yield can be high (>50%), and that it is boosted by high availability of nitrogen. We speculate that one partial cause of high fungal yield could be subsidy of fungal growth by dissolved organic carbon from outside decomposing leaves.