Lectin binding sites during Drosophila embryogenesis

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Ga-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.Dedicated to the memory of Jan Callaerts     

Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.

Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain-domain interactions lead to a rate-limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (kappa/IgG1). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late-folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native-like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like structure, follows a two-step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase

Gengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid-bound sialic acid exceeds that of the protein-bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.     

The effect of positive assortative mating at one locus on a second linked locus

Summary Considerations proceed from a model of positive assortative mating based on genotype at one locus, with an arbitrary number of alleles, assuming no selection, mutation, or migration, hypothetically infinite population size, and discrete non-overlapping generations. From these conditions, inferences are made about the genotypic structure at a linked locus, as well as about the corresponding 2-locus gametic structure.The following main results are presented: in the course of the generations, the genotypic structure at the second locus and the 2-locus gametic structure always tend to a limit responsive to the initial conditions concerning the joint genotypic structure at the two loci and the degree of assortativity and linkage. A complete, analytical representation of the limits is given. In particular, if assortative mating is only partial and at the same time linkage is not complete, a population is not able to maintain a permanent deviation of the gametic structure from linkage equilibrium, and thus the genotypic structure at the second locus tends to Hardy-Weinberg proportions. On the other hand, if initial linkage disequilibrium is combined with partial assortative mating and complete linkage (or with complete assortative mating and unlinked loci) the population maintains this disequilibrium and thus the genotypic structure at the second locus need not tend to Hardy-Weinberg proportions. It turns out that the conditions not only of complete linkage, but also of unlinked loci together with complete assortativity, imply no change in gametic structure from the initial structure.In order to demonstrate the influence of several parameters on the speed of convergence to and the magnitude of the respective limits, several graphs are included.     

Specific paracrystalline structures of rough endoplasmic reticulum in the follicular (stellate) cells of the dog adenohypophysis

Summary The fine structure of follicular cells of the adenohypophysis was examined in fetal, neonatal, and adult beagle dogs. Prior to birth, undifferentiated follicular cells are common. At birth mature cells that form follicles are routinely encountered. The fine structural appearance of follicular cells is unchanged between birth and adulthood. Follicular cells of puppies and adults are, however, distinguished by the presence of unusual complexes within distended cisternae of the rough endoplasmic reticulum. These complexes vary greatly in morphology, some appear as a maze of interconnecting tubules while others show a highly organized paracrystalline configuration. The presence of these paracrystalloid structures in follicular cells supports the view that they represent a distinct pituitary cell type.Supported by NIH Grants AM19743 and NS12969The authors wish to thank John Patrikes and Helen Mantulin for their expert technical assistance     

Short-term progressive early diagenesis in density bands of recent corals:Porites Colonies,Mauritius Island,Indian Ocean

Summary The growth history of some recentPorites colonies of Mauritius Island (Indian Ocean) was dated by sclerochronological methods. Couples of high-density and low-density bands represent the annual growth rate of the corals and allow the growth pattern of every year in the corallum to be counted. The growth and structure of the skeletons ofPorites solida andPorites lutea were investigated. Older parts of the aragonitic skeleton in these 10 to 20 year old corals show various secondary microstructures resulting from alterations and thickenings of the elements of the skeleton. The primary needle-like aragonite crystals are absent in older parts of the corallum and blocky aragonitic cements can occur. Pores and primary skeletal elements are overgrown by new microstructures. These microstructures are caused by secondary cementation and exhibit frontal zones (Stirnzonen), zigzag-like and pseudolamellar-structures. The lamellar structures can be compared with similar structures in the exoskeleton of some Rugosa. A very short early diagenesis within the recent corals is responsible for the thickening and alteration of skeletal elements. It occurs only 4 to 5 years after the formation of the skeleton and tends to increase in importance in older parts of the corallum. Nevertheless, there is no proof for any alteration of aragonite to calcite.     

Energetic approach to the folding of alpha/beta barrels

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.