Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

Voltage-gated sodium channels (Na_v) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na_v α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na_vs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na_vs.     

Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV

Kinetic studies of pig kidney dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5) were carried out using substrates possessing a side-chain of different length at the P2 position (or amino-terminal position in this case) such as Lys-, Arg-, Phe-, Met-, Ser-, His-, Glu- and Gly-Pro-pNA. The hydrolytic coefficient (Kcat/Km) has determined in the order Met- greater than Glu- greater than Ser- greater than His- greater than Phe- greater than Lys- greater than Gly- greater than Arg-, indicating a gradual increase with elongation of the side-chain from 0.03 to 0.60 nm followed by a decline when side-chain length approached 0.70 nm. Thus, the most probable depth of the side-chain pocket at the S2 subsite of the enzyme is proposed to be 0.50-0.60nm.     

Das elektronenmikroskopische Bild der Rattenspermien nach der Befruchtung

Zusammenfassung Aus der Tube von Ratten gewonnene Eier bzw. Frühstadien bis zu 4 Tagen nach der Befruchtung wurden elektronenmikroskopisch untersucht. Den eindringenden Mittelstücken fehlt eine begrenzende Membran. Sie bestehen aus einer peripheren Mitochondrienschraube und dem axialen Komplex mit den inneren Tubuli und neun breiten elektronendichten Säulen. Nach der ersten Furchungsteilung beginnen die Mitochondrien zu schwellen, verlieren Matrix und Cristae, lösen sich vom axialen Komplex und sind schließlich in Stadien mit mehr als 4 Zellen nicht mehr nachzuweisen. Nach der 2. Teilung lösen sich auch die Tubuli auf. Stücke der dicken Säulen kommen noch vereinzelt jenseits des 8-Zellstadiums vor. Eine Weitergabe von genetischem Material aus dem Cytoplasma über eine Teilung der Mitochondrien wird deshalb bestritten. Die Auflockerung des Spermienkopfes und die Bildung der Vorkerne werden beschrieben.

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Fine structure of spermatozoa of the rat after the entry into the egg

Summary Eggs isolated from the rat oviduct after fertilization have been studied with the electron microscope. The penetrating middle piece of the spermatozoon lacks a membrane. The mitochondria form a helical mitochondrial sheath around the axial complex with the central fibrils (9 + 2 pattern) and 9 electron dense outer fibers. The mitochondria begin to swell after the first cleavage. There is a considerable loss of matrix and the number of cristae decreases. Subsequently the mitochondria are discharged from the axial complex. When the ova have reached the 4-cell stage, mitochondria are no longer detectable. The central fibrils of the middle piece of the spermatozoa also disappear after the second division, but parts of the dense outer fibers may still be seen beyond the 16-cell stage. The results of this study do not favour the concept of a passage of genetic material from the sperm mitochondria to the egg cell. Division of sperm mitochondria certainly does not take place, but the actual fate of mitochondrial DNA has not been traced so far. The gradual loss of density of the sperm head and the formation of the pronucleus are described.

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Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Herrn Prof. Dr. Wilhelm Masshoff zum 60. Geburtstag gewidmet.     

Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Structure-activity relationship in the enzymatic hydrolysis of dipeptide aryl amides by dipeptidyl peptidase IV

Quantitative structure activity analysis of the substrate types Ala-Ala-AR and Ala-Pro-AR containing different substituents in the aryl ring showed that the rate-limiting step in the hydrolysis of the alanine substrates by dipeptidyl peptidase IV occurs in th acylation reaction (kcat approximately k2). Probably, the tetrahedral intermediate of the acylation process has a real life time. The positive q-value of the Hammett-equation in k'cat suggests that the N-atom of the arylamide is charged more negatively in the transition state TI not equal to than in the original state TI. The analysis of the quantitative conformation activity relationship (QCAR) gives information on the steric situation in the tetrahedral intermediate of the acylation step near the transition state. The rate limiting step in the hydrolysis of the substrates of the proline type occurs in the deacylation reaction.     

The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation.

The paramyxovirus nucleocapsid proteins (NPs) are relatively well conserved, except for the C-terminal 20% (or ca. 100 amino acids), referred to as the tail. We have examined whether this hypervariable tail is required for genome synthesis, both in vitro, where synthesis is predominantly from the input templates, and in vivo, where multiple rounds of amplification occur. In these viruses, genome synthesis and assembly of the nascent chain are coupled. We find that the tail is required in vivo but not in vitro. Closer examination of the in vivo system showed that the tailless NP could encapsidate the genome chain but that amplification did not occur. We interpret these results as indicating that the tail is not required for RNA assembly but is required for the template to function in RNA synthesis. Relatively small deletions within the conserved N-terminal 80% of the protein, on the other hand, rendered the protein nonfunctional in either system. The possible functions of the tail in RNA synthesis are discussed.     

Phylogenetic relationships within the aplocheiloid fish genus Rivulus (Cyprinodontiformes, Rivulidae): implications for Caribbean and Central American biogeography

We examined the phylogenetic relationships of 16 northern species of the aplocheiloid genus Rivulus inhabiting the Caribbean, Central America, and South America. A total of 714 base pairs per taxon were sequenced from two segments of the mitochondrial genome, 12S rRNA and cytochrome b. Both parsimony and neighbor-joining analyses suggest an ancient vicariant origin of the Greater Antillean taxa, in addition to a quite recent dispersal of species into the Lesser Antilles from the South American mainland. Combined analyses support the monophyly of the northern South American assemblage as the sister group of a Central American/Columbian biota. However, the monophyly of the Central American biota remains uncertain. Divergence estimates for the Central American taxa are calibrated from the Last Cretaceous separation of the proto-Antilles from the Americas. These data suggest that the extant Central American taxa represent the descendants of at least two separate invasions during the Cenozoic, prior to the closing of the Panamanian isthmus. Times are consistent with the extensive evidence for reptilian and mammalian exchange throughout the Cenozoic.      

Modeling residue usage in aligned protein sequences via maximum likelihood

A computational method is presented for characterizing residue usage, i.e., site-specific residue frequencies, in aligned protein sequences. The method obtains frequency estimates that maximize the likelihood of the sequences in a simple model for sequence evolution, given a tree or a set of candidate trees computed by other methods. These maximum- likelihood frequencies constitute a profile of the sequences, and thus the method offers a rigorous alternative to sequence weighting for constructing such a profile. The ability of this method to discard misleading phylogenetic effects allows the biochemical propensities of different positions in a sequence to be more clearly observed and interpreted.