Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation.

Genetic variations in dysbindin-1 (dystrobrevin-binding protein-1) are one of the most commonly reported variations associated with schizophrenia. As schizophrenia could be regarded as a neurodevelopmental disorder resulting from abnormalities of synaptic connectivity, we attempted to clarify the function of dysbindin-1 in neuronal development. We examined the developmental change of dysbindin-1 in rat brain by western blotting and found that a 50 kDa isoform is highly expressed during the embryonic stage, whereas a 40 kDa one is detected at postnatal day 11 and increased thereafter. Immunofluorescent analyses revealed that dysbindin-1 is enriched at the spine-like structure of primary cultured rat hippocampal neurons. We identified WAVE2, but not N-WASP, as a binding partner for dysbindin-1. We also found that Abi-1, a binding molecule for WAVE2 involved in spine morphogenesis, interacts with dysbindin-1. Although dysbindin-1, WAVE2 and Abi-1 form a ternary complex, dysbindin-1 promoted the binding of WAVE2 to Abi-1. RNA interference-mediated knockdown of dysbindin-1 led to the generation of abnormally elongated immature dendritic protrusions. The present results indicate possible functions of dysbindin-1 at the postsynapse in the regulation of dendritic spine morphogenesis through the interaction with WAVE2 and Abi-1.

Characterization of a vitellogenin gene reveals two phase regulation of vitellogenesis by engorgement and mating in the soft tick Ornithodoros moubata (Acari: Argasidae).

Synthesis of the precursor yolk protein vitellogenin (Vg) occurs after engorgement in haematophagous arthropods. We identified the Vg cDNA of the soft tick Ornithodoros moubata (OmVg) and compared its expression in mated and virgin females. Both mated and virgin females showed increases in OmVg expression after engorgement but expression was higher in mated females than virgin females particularly as time advanced. Delayed mating in virgin females induced an increase in OmVg expression. OmVg expression was observed in the midgut and fat body by whole mount in situ hybridization, but enlarged fat body with high expression occurred in only mated females during the late phase of vitellogenesis. Therefore, engorgement initially induces OmVg expression but mating is necessary for continued Vg expression to produce mature eggs.

Molecular characterization of a gene encoding juvenile hormone esterase in the red flour beetle, Tribolium castaneum.

Juvenile hormone esterases (JHEs) are required for the degradation of juvenile hormones (JHs) in insects. Here, we report the cloning and analysis of the jhe gene in the red flour beetle, Tribolium castaneum, a model insect of Coleoptera. The Tcjhe gene was strongly expressed at the final instar larva, as would be expected if it functioned to decrease the JH titer at this stage. A recombinant TcJHE protein efficiently degraded JH III, suggesting that the enzyme functions in vivo as a JH-specific degradation enzyme. This is the first report describing the developmental expression profile of the jhe gene whose enzymatic activity was shown in Coleoptera, and the new data reported here will aid elucidation of the mechanism of JH titer regulation in insects.

Macroglobulin structure: homology of mu and gamma heavy chains of human immunoglobulins.

The amino acid sequence of fragments obtained by cyanogen bromide cleavage of the mu-chain of a human gammaM-globulin is homologous to the NH(2)-terminal sequences of the gamma-chain of human and rabbit gammaG-globulins and is related to that of human light chains. This supports the hypothesis that light and heavy chains evolved from a common ancestral gene.

Immunoglobulin structure: variation in amino acid sequence and length of human lambda light chains.

Variation and conservation in the primary structure of human lambda light chains is revealed by complete amino acid sequence of three Bence Jones proteins. These proteins differ in amino acid sequence in from 38 to 48 positions; they are of unequal length in the amino-terminal half of the chain but have identical sequence in the last 105 amino acids.

Variation and homology in the mu and gamma heavy chains of human immunoglobulins.

Sequence analysis of an 1gM immunoglobulin shows that the variable regions of hunman micro and gamma1 heavy chainis may have twice as much homology as their constant regions and that evolutionary divergence of micro and gamma1 heavy chain genes occurred not long after the separation of heavy and light chain genes.

Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain.

Lzts1 controls both neuronal delamination and outer radial glial-like cell generation during mammalian cerebral development.

In the developing central nervous system, cell departure from the apical surface is the initial and fundamental step to form the 3D, organized architecture. Both delamination of differentiating cells and repositioning of progenitors to generate outer radial glial cells (oRGs) contribute to mammalian neocortical expansion; however, a comprehensive understanding of their mechanisms is lacking. Here, we demonstrate that Lzts1, a molecule associated with microtubule components, promotes both cell departure events. In neuronally committed cells, Lzts1 functions in apical delamination by altering apical junctional organization. In apical RGs (aRGs), Lzts1 expression is variable, depending on Hes1 expression levels. According to its differential levels, Lzts1 induces diverse RG behaviors: planar division, oblique divisions of aRGs that generate oRGs, and their mitotic somal translocation. Loss-of-function of lzts1 impairs all these cell departure processes. Thus, Lzts1 functions as a master modulator of cellular dynamics, contributing to increasing complexity of the cerebral architecture during evolution.

Diets with no or low amounts of dietary fiber can reduce small intestinal ulcers induced by non-steroidal anti-inflammatory drugs in dogs.

Recent progress in endoscopic techniques has revealed that non-steroidal anti-inflammatory drugs (NSAIDs) often cause ulcers in the small intestine in humans, but effective therapy is not available at present. In the present study, we investigated the effects of feeding condition and the amount of dietary fiber (DF) in the diet on the formation of gastrointestinal ulcers induced by NSAIDs in dogs. Several types of diets containing various percentages of DF were given to dogs. Indomethacin (1 or 3 mg/kg, p.o.), ketoprofen (2 mg/kg, s.c.), or fulnixin (1 mg/kg, s.c.) was administered once daily at 10 a.m. after a morning meal or without a morning meal (fasted condition) for 3 - 7 days. Gastrointestinal lesions were examined 24 h after the final dose of the drugs. When indomethacin (3 mg/kg) was administered after a morning meal (fed condition) for 7 days, it produced many lesions in the small intestine. However, when it was given in the fasted condition without the morning meal, the lesions were markedly decreased. All the NSAIDs given after feeding of regular dry food containing 6% DF once a day for 3 days produced many lesions in the small intestine. The lesions were decreased or increased in dogs given prescription diets containing low DF (1.1%) and high DF (15.4%), respectively. Furthermore, lesions were not observed in dogs given canned diet containing very low DF (< 0.1%), whereas lesions appeared again in dogs given canned diet supplemented with cellulose (3 or 10%) but not with pectin (10%). These results suggested that both feeding condition and insoluble DF, such as cellulose in the diet, play an important role in the formation of NSAID-induced small intestinal lesions, and that a diet with no or low amounts of DF may decrease gastrointestinal side-effects associated with the use of NSAIDs.

Metabolism of 3-deoxyglucosone, an intermediate compound in the Maillard reaction, administered orally or intravenously to rats.

The Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [14C]3DG was synthesized from [14C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [14C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO2 were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [14C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO2 were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [14C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and 13C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.

Renal handling of urate in the syndrome of inappropriate secretion of antidiuretic hormone.

Three patients with the syndrome of inappropriate secretion of antidiuretic hormone had elevated uric acid clearances. Their uric acid clearances decreased markedly after the administration of pyrazinamide. Probenecid was given to two of them and it produced large increases in uric acid clearance. These data suggest that enhanced secretion in the renal tubules was responsible for the increased clearance of uric acid. This article provides evidence that hypouricemia in the syndrome of inappropriate secretion of antidiuretic hormone is due to increased tubular urate secretion.

Immunochemical determinant and serological specificity of Candida krusei.

We have investigated the chemical structure of the antigenic determinant of Candida krusei cell-wall mannan. Acetolysis of the mannan, obtained by extraction with alkali and purified as copper complex, gave five oligosaccharides (from mono- to pentasaccharide) and a minor amount of an octasaccharide. Partial acetolysis of the mannan gave a large amount of mannooctaose. We have examined the inhibition by these oligosaccharides of the precipitin reaction between anti-Candida krusei serum and homologous mannan, and found that the mannooctaose was the most effective inhibitor. Results obtained by proton magnetic resonance spectroscopy, methylation analysis, and other structural studies of Candida krusei mannan, suggest that the octasaccharide possesses six alpha (1-2) linkages and one alpha (1-6) linkage located in the middle of the chain, and that this mannooctaose may be responsible for the specificity of Candida krusei cell-wall mannan.

Immunological characterization and structural studies of normal fecal antigen-1 related to carcinoembryonic antigen.

Normal fecal antigen-1 (NFA-1), which is a carcinoembryonic antigen (CEA)-related glycoprotein with a mol. wt of 20,000-30,000, was purified from normal adult feces by immuno-adsorption, gel filtration and ion exchange chromatography. Highly purified NFA-1 was partially cross-reactive with CEA but antigenically unrelated to nonspecific cross-reacting antigen (NCA) which was also cross-reactive with CEA. NFA-1 also had a unique determinant not present in CEA or other related antigens including NCA. In a solid-phase RIA system, the reactivity of NFA-1 with a specific anti-CEA antiserum was much stronger than that of NCA. Although digestion with Pronase E did not affect the antigenicity of NFA-1, reduction and alkylation destroyed its antigenic reactivity. The total amount of carbohydrate in NFA-1 was 13.3%, compared to 52.4% in CEA and 21.6% in NCA. The amino acid composition of NFA-1 was similar to that of CEA. The sequence of the first 10 NH2-terminal amino acids in NFA-1 was Ala-Glu-Pro-Pro-Lys-Pro-Phe-Ile-(Thr)-Ser. This was totally different from that of the first NH2-terminal amino acids of CEA isolated from tumor tissue.

Further comparative studies on chemical properties of carcinoembryonic antigen in tumor tissues and closely related antigens in adult feces and meconium.

The chemical structure of carcinoembryonic antigen (CEA) and two closely related antigens, normal fecal antigen-2 (NFA-2) in normal adult feces and nonspecific cross-reacting antigen-2 (NCA-2) in the meconium, were further analyzed comparatively. The NH2-terminal amino acid sequence of NCA-2 was newly determined to position 18 and found to be identical to that so far determined for CEA- and NFA-2. After proteolytic digestion with chymotrypsin or protease V8, the digests of these antigens showed two groups of fragments upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One consisted of the sharply banded fragments which were identical in all antigens and stained only with Coomassie brilliant blue (CBB) (five bands in the range 2500-10,000 daltons for chymotrypsin and 11 bands in the range 8000-35,000 daltons for protease V8, respectively), and the other consisted of the dispersed fragments which had variable mol. wts in the range 10,000-100,000 and were stainable with both CBB and periodic acid-Schiff reagent. Elution profiles of CEA, NFA-2, and NCA-2 from lectin columns, especially from concanavalin A-Sepharose columns, suggested some differences in oligosaccharide chains between them. These results indicate that the fundamental chemical structure of these antigens seems to be very similar to one another and is divided into two parts; an homologous portion(s) which is common to all three antigens and contains no sialylated sugar components, and a heterogeneous portion(s) which is variable among these antigens and contains sialylated sugar components.

[Yolk sac tumor of the anterior mediastinum and pulmonary metastases; report of a case].

A 31-year-old female was clinically diagnosed as having a anterior mediastinal yolk sac tumor because of the elevation of the AFP (17,500 ng/ml), a large mass lesion (9 x 5 cm) in the anterior mediastinum and bilateral lung metastases. After 4 courses of chemotherapy with cisplatin (CDDP), etoposide (VP-16) and bleomycin hydrochloride (BLM), the mediastinal mass reduced in size significantly and the serum AFP level reached within normal range. Fluorodeoxyglucose-positron emission tomography (FDG-PET) showed a weak uptake in the mediastinum, accordingly the operation was performed. The tumor was completely removed and there were no viable foci of the tumor in part of the tumor. After the operation, 4 courses of chemotherapy with carboplatin (CBDCA), VP-16 and ifosfamide (IFM) were performed. She is alive without evidence of recurrence in 5 months after operation. It was noticed that the serum AFP is a useful indicator for determing the chance of operation after chemotherapy.

In vitro processing of amyloid precursor protein by cathepsin D.

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.

Amino acid sequence of an amyloidogenic Bence Jones protein in myeloma-associated systemic amyloidosis.

The complete amino acid sequence of an amyloidogenic Bence Jones protein (NIG-84) from an individual with myeloma-associated systemic amyloidosis has been determined. The protein, with a blocked N-terminus, represents a complete light chain consisting of 217 residues and it has a structural feature characteristic of the V lambda II subgroup. In addition to a two-residue insertion at positions 28 and 29, it has an additional rare insertion of alanine at position 100. NIG-84 is an example of the first complete sequence presented for the amyloidogenic Bence Jones protein of the V lambda II subgroup.

Antibodies against defined carbohydrate structures of Candida albicans protect H9 cells against infection with human immunodeficiency virus-1 in vitro.

In this study we raised antibodies against Candida albicans mannans of serotype A and B which comprise mannose alpha(1----2)-and mannose alpha(1----3)-linked residues. These antibodies inhibited human immunodeficiency virus type IIIB (HIV-IIIB) infection of H9 cells in vitro; 5 micrograms/ml of antibodies against mannan from serotype A and 10 micrograms/ml of antibodies against serotype B mannan were sufficient to inhibit infection by almost 100 and 85%, respectively, after an incubation period of 4 days. During a prolonged incubation period (8-12 days), the amount of HIV particles (as measured by reverse transcriptase activity in the culture medium) increased again in assays with antibodies raised against serotype B, but only little in assays containing antibodies against serotype A. Applying the Western blotting technique and a novel enzyme-linked immunosorbent assay system it was established that the antibodies reacted with the gp120 of HIV-1 exclusively. Immunofluorescence inspection using a confocal laser scanning microscope revealed that the gp120 protein is exposed on the outer surface of H9 cells where it is recognized by the anti-mannan antibodies. These results indicate that mannan residues of C. albicans can serve as antigens to raise neutralizing antibodies against HIV infection.