Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll? gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg²⁺ and Ca²⁺ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg²⁺ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.     

Spontaneous and Engineered Deletions in the 3′ Noncoding Region of Tick-Borne Encephalitis Virus: Construction of Highly Attenuated Mutants of a Flavivirus

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3′ NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3′-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3′ NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3′ NCR and never extended into the conserved 3′-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3′-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.The genus Flavivirus (family Flaviviridae) consists of small, enveloped, mainly mosquito- or tick-transmitted viruses with an unsegmented positive-stranded RNA genome (34). Some of these viruses are human pathogens of global medical importance, most notably yellow fever virus, the dengue (DEN) viruses, Japanese encephalitis virus, and tick-borne encephalitis (TBE) virus (22). In spite of the availability of vaccines against several of these viruses, flavivirus infections continue to be a major health problem in many countries of the world. Elucidation of the molecular basis of the pathogenicity of these viruses and identification of specific determinants of virulence are therefore a major focus of flavivirus research.The approximately 11-kb flavivirus genome (for a review, see reference 3) encodes three structural proteins (the capsid protein C, the small membrane protein M, which is formed by proteolytic cleavage from its precursor protein prM, and the large envelope protein E) and seven nonstructural proteins (the glycoprotein NS1, the protease component NS2A, NS2B, the protease/helicase NS3, NS4A, NS4B, and the RNA polymerase NS5). All of the viral proteins are encoded within a single long open reading frame which is flanked by noncoding regions (NCR) believed to carry regulatory elements involved in replication, translation, and packaging of the genome. Molecular analyses of natural low-virulence strains and strains attenuated in vitro by passaging procedures or, more recently, by specific mutagenesis techniques, have shown that genetic determinants that govern the virulence of flaviviruses are located within the coding regions of both structural and nonstructural proteins as well as within the flanking NCRs (2, 21, 26; for reviews, see references 20 and 22). In this study, we focus on the effects of deletions in the 3′ noncoding region (3′ NCR) of TBE virus.TBE virus causes widespread human disease in many European and Asian countries, and its molecular biology has been studied in some detail (29; for a review, see reference 9). The length of the 3′ NCR of TBE virus was previously found to be remarkably heterogeneous even among closely related strains, ranging from approximately 450 to almost 800 nucleotides (31). A more detailed analysis indicated that the 3′ NCR can be divided into a 3′-terminal core element of approximately 340 nucleotides in length and a variable region located between the core element and the open reading frame. The core element is present in all strains investigated so far, and its nucleotide sequence is highly conserved among strains. The entire core element is predicted to fold into a well-defined secondary structure independent of the sequence of the adjacent variable genomic element (27). The variable region is characterized by low sequence conservation, extensive size variability between strains, repetitive sequence elements, and an internal poly(A) tract in certain TBE virus strains (15, 31). Evidence for 3′-NCR size heterogeneity and specific RNA-folding patterns for the 3′-terminal approximately 400 nucleotides have also been observed with several other flaviviruses (5, 24, 25, 33). A similar organization of the 3′ NCRs also appears to be shared by members of the other two genera of the family Flaviviridae, pestiviruses and hepaciviruses (13, 23, 30, 35).Although the functional importance of the flavivirus 3′ NCR is generally acknowledged, the assumed involvement of particular sequence elements in replication, modulation of translation, or packaging is largely hypothetical. Evidence for functionality is so far based mostly on the identification of highly conserved RNA sequence elements or folding patterns by computer techniques. A few studies have provided direct evidence for the binding of protein factors to the stem-loop structure closest to the 3′ terminus (1, 4). Moreover, Men et al. (21) demonstrated that certain deletions introduced into the 3′ NCR of DEN-4 virus resulted in viable mutants with significantly restricted growth properties. By this approach, these researchers were able to identify particular sequences that are required for viability and others that can be deleted without apparent impact on the biology of DEN-4 virus. Studying replicons of Kunjin virus, Khromykh and Westaway (14) found that parts of the 3′ NCR could be deleted or even replaced by a foreign protein expression cassette without loss of replication competence. The 3′-NCR sequences of these flaviviruses, however, exhibit very little homology to the sequences of the tick-borne flaviviruses, which even lack the sequence boxes CS1 and CS2 that are conserved among all mosquito-borne flaviviruses (7, 16).The establishment of an efficient and stable infectious cDNA clone system for TBE virus European subtype prototypic strain Neudoerfl (17) has enabled us to test the functional importance of 3′-NCR sequence elements of this virus by a directed mutagenesis approach. As reported in this communication, spontaneous deletions in the variable region of strain Neudoerfl occur frequently during viral growth in cell culture or in infected animals. This prompted us to construct 3′-NCR deletion mutants of variable lengths to study the influence of these deletions on the biological properties of TBE virus. Our results demonstrate a correlation between the presence of certain RNA sequences or secondary structures and growth properties, viability, and attenuation of the resulting virus mutants. We present several 3′-NCR deletion mutants that are 4 orders of magnitude less virulent than wild-type TBE virus.With regard to vaccine development, the most desirable mutations are ones that are genetically stable and cause significant attenuation but maintain adequate replication properties in cell culture and strong immunogenicity in animals even at low inoculation doses. The evaluation of the TBE virus mutants presented in this article indicates that certain deletions in the 3′ NCR can indeed meet these criteria.     

Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity

Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic compounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)-O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)-O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein. No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the formation of the transient intermediate and for substrate turnover.     

Mutations in microcephalin cause aberrant regulation of chromosome condensation

Microcephalin (MCPH1) is a gene mutated in primary microcephaly, an autosomal recessive neurodevelopmental disorder in which there is a marked reduction in brain size. PCC syndrome is a recently described disorder of microcephaly, short stature, and misregulated chromosome condensation. Here, we report the finding that MCPH1 primary microcephaly and PCC syndrome are allelic disorders, both having mutations in the MCPH1 gene. The two conditions share a common cellular phenotype of premature chromosome condensation in the early G2 phase of the cell cycle, which, therefore, appears to be a useful diagnostic marker for individuals with MCPH1 gene mutations. We demonstrate that an siRNA-mediated depletion of MCPH1 is sufficient to reproduce this phenotype and also show that MCPH1-deficient cells exhibit delayed decondensation postmitosis. These findings implicate microcephalin as a novel regulator of chromosome condensation and link the apparently disparate fields of neurogenesis and chromosome biology. Further characterization of MCPH1 is thus likely to lead to fundamental insights into both the regulation of chromosome condensation and neurodevelopment.     

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G₂/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.Key words: chromosome condensation, DNA damage, G₂/M checkpoint, ionizing radiation, PCC syndrome, primary microcephaly, repair foci     

Amorphophallus: New insights into pollen morphology and the chemical nature of the pollen wall

In pollen characters, Amorphophallus is one of the most diverse genera in the Araceae. The present work is a critical survey of contradicting reports on the impact of acetolysis treatment on Amorphophallus pollen, on the chemical nature of the outer pollen wall layer and of electron-dense (dark) granules found within it. Furthermore, we wanted to clarify the pollen polarity and to test conclusions based on different preparation techniques. Pollen morphology of 25 species is investigated by light microscopy, scanning electron microscopy and transmission electron microscopy. Our results show that Amorphophallus pollen is not resistant to acetolysis treatment. The use of different transmission electron microscopy staining methods proved the polysaccharide nature of the outer pollen wall layer and of the granules within it. Moreover, an additional thin surface layer was found in all investigated species. Microspores in early and late tetrad stages show that the less convex side of the microspore is the proximal face and the more convex side the distal face. The extrusion of pollen in strands is illustrated for the first time by light microscopy and scanning electron microscopy. Furthermore, observations of pollen in water showed that in some of the investigated species the pollen wall is shed immediately before pollen tube formation.