Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal "clip" domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.     

Molecular characterization and expression analysis of Manduca sexta allatotropin

Juvenile hormones (JH) are a class of regulatory sesquiterpenoids that control metamorphosis in immature insects and reproduction in adult insects. The regulation of JH synthesis by the corpora allata (CA), a pair of endocrine glands with nervous connections to the brain, is achieved by a complex interplay of stimulatory and inhibitory factors mediated in part by the brain. The neuropeptide, allatotropin (Mas AT), was recently isolated and sequenced from the brain of the tobacco hornworm Manduca sexta. Mas AT is a 13-residue amidated peptide that activates JH synthesis in adult, but not larval, lepidopteran CA. A 23-nucleotide degenerate oligonucleotide was designed based on the peptide sequence and was used to isolate the Mas AT genomic clone. The Mas AT gene is expressed as three mRNAs which differ from one another by alternative splicing. These mRNAs are predicted to encode three distinct prohormones, each containing Mas AT. A restriction fragment from the genomic clone was then used to isolate the cDNA clone. In situ hybridization and immunohistochemistry studies show that Mas AT is expressed in both the central and enteric nervous systems. Cells expressing Mas AT were identified in the brain, frontal ganglion and terminal ganglion.     

Molecular cloning, cDNA sequence analysis, and chromosomal localization of mouse Pkd2

The gene responsible for the second form of autosomal dominant polycystic kidney disease, PKD2, has recently been identified. We now describe the cloning, genomic localization, cDNA sequence, and expression analysis of its murine homologue, Pkd2. The cloned cDNA sequence is 5134 bp long and is predicted to encode a 966-amino-acid integral membrane protein with six membrane-spanning domains and intracellular NH2 and COOH termini. Pkd2 is highly conserved with 91% identity and 98% similarity to polycystin-2 at the amino acid level. Pkd2 mRNA is widely expressed in mouse tissues. Pkd2 maps to mouse Chromosome 5 and is excluded as a candidate gene for previously mapped mouse mutations resulting in a polycystic kidney phenotype.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Palaeontology provides the only direct record for morphological and genetic change through time and uniquely contributes to systematics in two ways: by providing access to denser taxon sampling than is otherwise possible and by dating divergence times. Claims that ancient DNA has survived millions of years in certain fossils suggested the possibility that palaeontology could contribute directly to molecular systematic studies. Unfortunately, none of the supposed geologically ancient DNA records stands up to detailed scrutiny and fossils therefore contribute primarily through the morphological information they preserve. Denser taxon sampling can improve the accuracy of phylogenetic estimates primarily through allowing better discrimination of homoplasy from homology. This in turn leads to more accurate hypotheses of character transformation. Denser taxon sampling also offers the opportunity for more accurate rooting, since more characters can be polarized by reference to a stem-group taxon than to an extant sister-group taxon. Missing data can be a problem for fossils, but is not crippling. Finally the temporal order of clade appearances in the fossil record can provide ancillary evidence for selecting a working phylogeny from among a number of equally most parsimonious cladograms.     

Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed.     

Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated (Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.     

Molecular cloning of a cDNA from Brassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a lambda gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis of Brassica napus genomic DNA revealed the presence of 6 genes, 3 derived from the Brassica rapa parent and 3 from Brassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The 3-years-old patient presents bilateral exophthalmia and papillar stasis at both eyes. The cranial examination shows the frontal boss at the level of the anterior fontanelle and the hypoplasia of the inferior facial floor. The cranial radiography shows digital impressions on the entire projection surface of the cap. The child also presents mental retardation. The affection integrates in the category of craniofacial dysostosis described by Crouzon, maladies which are transmitted autosomal with great penetrance.     

Molecular cloning of a canine metallothionein cDNA

A canine metallothionein cDNA obtained from the liver of a cadmium-treated beagle was cloned and sequenced. Asn at position 4 conserved among all mammalian metallothionein-1 and metallothionein-2 is replaced by Asp in the canine metallothionein cDNA clone. Because the acidic amino acid doesn't exist at either position 10 or 11 in the deduced amino acid sequence, it is supposed that this cDNA is derived from canine metallothionein-1 mRNA. Northern blot analysis using the cDNA as a probe revealed the induction of the canine metallothionein mRNA expression in the liver and kidney of a cadmium-treated beagle. Thus, the canine metallothionein cDNA obtained in the present study should provide an useful tool for the molecular investigation of metallothionein in dog.     

Partial characterization of allatinhibin, a neurohormone of Manduca sexta

During the last larval stage, corpora allata (CA) of Manduca sexta are inactivated by a factor from the brain. Apparently the same factor (allatinhibin, AI) is secreted by day 4 Vth instar brains kept overnight in Grace's medium. AI is rapidly inactivated by heat or acid but withstands exposure to alkali and can be recovered after freezing and lyophilization. Exposure to pronase, chymotrypsin, carboxypeptidases-A and -Y, as well as leucine aminopeptidase eliminated AI activity completely, whereas after exposure to trypsin and protease XVII-S, some residual activity remained. Inactivation by pyroglutamate aminopeptidase is interpreted as being due to prolinase activity of this enzyme. Incubation of CA with gentamicin, an aminoglycoside antibiotic, affects neither their ability to produce JH in vitro nor their viability in implantation assays. However, AI did not inactivate CA in the presence of low concentrations of gentamicin. This effect was used to guard against false positive assay results possibly produced by allatotoxic contamination. AI was purified by chromatography on Sephadex G-25. All activity recovered emerged from the columns in intermediate fractions with an apparent M(r) of 1,000-2,000.     

Alx-4: cDNA cloning and characterization of a novel paired-type homeodomain protein

Fish react to handling and capture with a burst of exercise that affects them deeply. The present study examines the effect of such severe exercise and the time course of recovery on the hematology (including spleen response) and metabolism of a population of cultured rainbow trout. Exercise was induced by continuous chasing for 5 min when the trout showed signs of exhaustion. Such exercise led to spleen contraction and an increase in haematocrit values. Carbohydrates were mobilized and anaerobic glycolysis produced lactate without significant effect on lipid metabolism. The conclusion is reached that the respiratory properties of rainbow trout blood do not change following severe exercise, while muscle anaerobic metabolism is slightly activated as deduced from the fast and short lactacidemia observed, which may have been related to a reduced stressing component, as the exercise was performed in the same environment in which the fish were reared.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

Molecular cloning of a murine cDNA encoding a novel protein, p38-2G4, which varies with the cell cycle

Proliferating cells express genes active in cell cycle control. The modulation of control genes and factors are required to maintain critical cell cycle activities. We used a set of monoclonal antibodies prepared against DNA-binding proteins from Ehrlich ascites tumor cells in immunofluorescent microscopy to screen for proteins showing cell cycle-specific staining patterns. Here, we report cloning and characterizing of a novel mitogen-inducible gene from murine macrophages that predicts a cell cycle-specifically modulated nuclear protein of 38 kDa, designated p38-2G4. p38-2G4 displayed a speckled pattern of varying fluorescence intensity confined to the nucleus, but sparing the nucleoli. Strongly stained granules were observed between G1 and mid S phase, followed by a less abundant punctated arrangement toward the end of S phase, and negative fluorescence at the S/G2 transition. Thereafter, the nuclear staining reappeared. Additionally, p38-2G4 expression vanished in G0-arrested cells and was restored after release from growth arrest. p38-2G4 conserved in vertebrates by means of immunofluorescence data contains a number of putative phosphorylation sites, a cryptic nuclear localization signal, and an amphipathic helical domain. Our cDNA and its deduced amino acid sequence is related to a Schizosaccharomyces pombe gene encoding a 42-kDa protein that associates with curved DNA, suggesting that we have cloned the murine homologue of the S. pombe gene which defines a novel cell cycle-specifically modified and proliferation-associated nuclear protein in mammals.     

Molecular cloning of a mutated HOXB7 cDNA encoding a truncated transactivating homeodomain-containing protein

Bronchial infections are common in smokers and seem to be related to the presence of chronic bronchitis (CB). Why only some smokers develop repeated bronchial infections is not known. The aim of this study was to screen for immunological changes associated with disease in patients with CB and recurrent infectious exacerbations compared to asymptomatic smokers. Sixteen smokers with stable CB and recurrent infectious exacerbations, and 18 asymptomatic smokers, all without any immunomodulating treatment, underwent bronchoscopy and bronchoalveolar lavage (BAL). Smoking history and current smoking status were comparable. Serum levels of immunoglobulin (Ig)A, IgM, IgG and IgG subclasses were measured. Blood and BAL lymphocyte phenotypes and proliferative responses of peripheral blood mononuclear cells (PBMCs) to various stimulators were analysed. Unstimulated and tetanus toxoid-stimulated production of cytokines in PBMC cultures was measured. Natural killer (NK-) cell activity was analysed. A significantly (p<0.05) lower level of IgG3 was found in the CB group, and a significantly (p<0.01) higher proliferative response of PBMCs was found in the CB group after stimulation with diphtheria toxoid. Detectable levels of interleukin (IL)-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma, but not of IL-2, IL-4 or transforming growth factor-beta2, were found in supernatants from cultured cells in both study groups. Stimulated TNF-alpha production was significantly (p<0.05) higher in the CB group. NK-cell activity did not differ significantly between the study groups. There were no major differences between the groups in lymphocyte subpopulations in blood or BAL. In conclusion, no major alterations in the analysed indices of cell-mediated and humoral immunity were found in patients with chronic bronchitis prone to recurrent infectious exacerbations when compared with asymptomatic smoking controls.     

Molecular cloning, cDNA sequence, and chromosomal assignment of the human radixin gene and two dispersed pseudogenes

Radixin is a cytoskeletal protein that may be important in linking actin to the plasma membrane. Recent cloning of the murine and porcine radixin cDNAs revealed a protein highly homologous to ezrin and moesin. We have cloned and sequenced the human radixin cDNA and found the predicted amino acid sequence for the human protein to be nearly identical to those predicted for radixin in the two other species. By Southern analyses of Chinese hamster x human somatic cell hybrid DNA and of PCR products derived from hybrids, the coding gene (RDX) was mapped to 11q. Fluorescence chromosomal in situ hybridization with a cDNA plasmid further localized this gene to band 11q23. However, PCR amplification with "radixin-specific" primers on the hybrid DNA panel yielded an additional, very similar DNA sequence that was further characterized by direct sequencing of PCR products. This sequence represents a truncated version and the respective locus (RDXP2) was assigned to Xp21.3. Furthermore, by employing a different set of primers, a third sequence was found that was 90% identical to the radixin sequence but contained termination codons and seemed to lack introns. This pseudogene (RDXP1) was mapped to 11p by Southern and PCR analyses.     

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.     

Molecular cloning and characterization of a highly conserved chicken cellular nucleic acid binding protein cDNA

Three variants of the Candida antarctica B lipase have been constructed and characterized. The variant containing the T103G mutation, which introduces the consensus sequence G-X-S-X-G found in most other known lipases, shows an increased thermostability but retains only half the specific activity of the native enzyme. Also in ester synthesis the activity is lowered but the specificity and enantioselectivity remains unchanged. The W104H mutant, in which more space is introduced into the active site, has more dramatically changed properties. Both the thermostability and the specific activity are slightly reduced but the activity and specificity in ester synthesis is highly different from the native enzyme. In general, the activity is very low and the enantioselectivity is, furthermore, highly reduced. Finally, the mutation M72L was introduced to increase the oxidation stability of the enzyme. This variant did exhibit an increased resistance towards oxidation but the thermostability was, unfortunately, also reduced.