The highly conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation.

The prion protein (PrP)-known for its central role in transmissible spongiform encephalopathies-has been reported to possess two nuclear localization signals and localize in the nuclei of certain cells in various forms. Although these data are superficially contradictory, it is apparent that nuclear forms of the prion protein can be found in cells in either the healthy or the diseased state. Here we report that Shadoo (Sho)-a member of the prion protein superfamily-is also found in the nucleus of several neural and non-neural cell lines as visualized by using an YFP-Sho construct. This nuclear localization is mediated by the (25-61) fragment of mouse Sho encompassing an (RXXX)8 motif. Bioinformatic analysis shows that the (RXXX)n motif (n=7-8) is a highly conserved and characteristic part of mammalian Shadoo proteins. Experiments to assess if Sho enters the nucleus by facilitated transport gave no decisive results: the inhibition of active processes that require energy in the cell, abolishes nuclear but not nucleolar accumulation. However, the (RXXX)8 motif is not able to mediate the nuclear transport of large fusion constructs exceeding the size limit of the nuclear pore for passive entry. Tracing the journey of various forms of Sho from translation to the nucleus and discerning the potential nuclear function of PrP and Sho requires further studies.

The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c, regulates microtubule binding and stability.

Phosphorylation of microtubule-associated protein 2 (MAP2) has a profound effect on microtubule stability and organization. In this work a consensus protein kinase A (PKA) phosphorylation site, T(220), of juvenile MAP2c is characterized. As confirmed by mass spectrometry, this site can be phosphorylated by PKA but shows less than average reactivity among the 3.5 +/- 0.5 phosphate residues incorporated into the protein. In contrast, T(220) is uniquely sensitive to dephosphorylation: three major Ser/Thr protein phosphatases, in the order of efficiency PP2B > PP2A(c) > PP1(c), remove this phosphate group first. MAP2c specifically dephosphorylated at this site binds and stabilizes microtubules stronger than either fully phosphorylated or nonphosphorylated MAP2c. Phosphorylation of this site also affects proteolytic sensitivity of MAP2c, which might represent a further level of control in this system. Thus, the phosphorylation state of T(220) may be a primary determinant of microtubule function.

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Dissecting insect development: baculovirus-mediated gene silencing in insects.

A novel concept applying baculovirus-mediated gene silencing to study insect gene function and regulation is described in this paper. A recombinant baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), was constructed with the juvenile hormone esterase (JHE) gene from the tobacco budworm Heliothis virescens in the antisense orientation, driven by the viral p10 promoter. Infection with this recombinant greatly reduced the haemolymph JHE level and resulted in aberrant morphogenesis of final-instar H. virescens larvae. The body organization remained larval, although the cuticle became hard and brown, similar to pupal cuticle. These results demonstrated that baculovirus-mediated gene silencing can be accomplished and utilized to dissect insect development and to design a new class of baculovirus insecticides.

Recombination of baculovirus DNA following lipofection of insect larvae.

By injection of a liposomal complex containing baculovirus DNA directly into the hemocoel of insect larvae a polyhedrosis disease was induced. After cotransfection of insect larvae with circular viral DNA and transfer vector DNA recombinant viruses were generated with a frequency of about 2%, similar to what is obtained in vitro using insect cell cultures. Based on these results an alternative strategy for the generation of recombinants can be derived for baculoviruses for which susceptible cell lines are not readily available. This strategy involves the injection of baculovirus DNA into susceptible larvae followed by in vivo cloning.

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo.

The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.

Purification of a new restriction endonuclease from Streptococcus mutans and identification of its recognition sequence.

SmuE I, a type II restriction endonuclease, has been isolated from Streptococcus mutans serotype E, which is an isoschizomes of Ava II recognizes the palidromic pentanucleotide sequence 5' GG/W/CC 3'. Similarly to Ava II, SmuE I cleaves the sequence, G--G/W/CC, generating 5' protruding fragment termini.

Effective removal of excess Coomassie blue from polyacrylamide gels.

The destaining of Coomassie Brilliant Blue-stained polyacrylamide gels usually is time consuming and requires large volumes of destaining solution. Removal of the Coomassie blue stain from the gel can be accomplished efficiently and economically by liquid-liquid extraction using a biphasic mixture of 1-butanol and 5% aqueous acetic acid.

Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labeling.

Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II [epoxy[3H]bishomofarnesyl diazoacetate ([3H]EBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)], and of the JH analog methoprene [[3H]methoprene diazoketone ([3H]MDK)] were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.

Fluorography of tritium-labeled proteins in silver-stained polyacrylamide gels.

Silver-staining of polyacrylamide gel electrophoresis (PAGE)-separated proteins allows sensitive detection of proteins but severely reduces the ability to detect weak beta-emitters present in the protein band. A simple procedure is described in which silver can be removed from a silver-stained PAGE gel (deargentation) using photographic fixer, and the silver-free gel can be enhanced and used for fluorography. A quantitative study of sensitivity is reported for 3H-labeled bovine serum albumin with a one-dimensional sodium dodecyl sulfate-PAGE slab gel.

Detection and microsequencing of juvenile hormone-binding proteins of an insect by the use of an iodinated juvenile hormone analog.

An [125I]iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high-affinity, (JH)-specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two-dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I-labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound gamma-emitter can be found by exposure of the dried gel or the electroblot to X-ray film. The radiolabeled protein spot can be excised from the Coomassie-stained glass fiber paper and subjected directly to gas-phase N-terminal amino acid sequencing. This non-destructive, non-denaturing technique may have wide applicability in identifying and sequencing ligand-specific binding proteins in complex mixtures.