Effects of photoperiod, temperature and aging on adult diapause termination and post-diapause development in female Asian comma butterflies, Polygonia c-aureum Linnaeus (Lepidoptera: Nymphalidae).

Polygonia c-aureum females exhibit photoperiodically induced imaginal diapause, characterized by cessation of ovarian development. Females grown at a short daylength (SD) entered imaginal diapause, whereas those grown at a long daylength (LD) produced eggs rapidly after adult emergence at 21 °C. The termination of diapause was influenced by daylength: diapause ended faster at LD than SD. Complete termination of diapause took 30 days in unchilled females reared under LD at 21 °C. On the other hand, prompt, synchronized and strong diapause termination occurred at post-chilling periods. Photoperiods at post-chilling periods affected ovarian development, when the length of pre-chilling periods or the length of chilling periods was shorter, suggesting that these treatments were not enough to complete diapause development. Ovarian development proceeded earlier in chilled and subsequent warmed females than unchilled females. Wing damage was remarkable at post-chilling periods when females were reared under an adequate length of pre-chilling and chilling periods, especially comparing with females under pre-overwintering conditions without chilling, indicating that post-diapause reproductive development was weak in unchilled females. Thus, exposure to low temperatures is necessary for a strong diapause termination in this butterfly.

Effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on reproduction in Polygonia c-aureum (Lepidoptera: Nymphalidae) in relation to adult diapause.

We investigated the effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on female and male reproduction in a nymphalid butterfly, Polygonia c-aureum. This butterfly has a facultative adult diapause controlled by the corpora allata and brain. Methoprene seems to terminate reproductive diapause, although transplantation experiments indicate that the activity of the corpora allata does not affect male mating behavior Endo (Dev Growth Differ 15:1-10, 1973a), suggesting that the brain may be involved in diapause. We found that exposure to methoprene promoted the development of ovaries and of the male accessory glands and simplex. On the other hand, exposure to 20-hydroxyecdysone did not promote the development of female and male reproductive organs and eupyrene sperm movement from the testis to the duplex in the adult stage. Ecdysteroid titer in both sexes was consistently low in adults. These results suggest that imaginal diapause is largely regulated by juvenile hormone in this butterfly.

Nucleotide sequence and chromosomal localization of the gene for pierisin-1, a DNA ADP-ribosylating protein, in the cabbage butterfly Pieris rapae.

Cabbage butterfly, Pieris rapae, contains a unique DNA ADP-ribosylating protein, pierisin-1, which transfers ADP-ribose moiety of NAD to guanine bases of DNA. Pierisin-like proteins are only distributed in subtribes Pierina, Aporiina and Appiadina of the family Pieridae. In this study, we obtained genomic clones carrying the pierisin-1 gene from adult samples of P. rapae by plaque hybridization. The pierisin-1 gene was found to consist of two exons, 0.1-kb exon 1 and 3.9-kb exon 2, and a 2.3-kb intron. In addition, we could demonstrate that the putative promoter in the about 3-kb upstream region from the transcription start site of the gene include a transcriptional activating motif involved in immune pathways and hormonal regulation. We also examined chromosomal localization of the pierisin-1 gene. Fluorescence in situ hybridization (FISH) analysis using Cy3-labeled pierisin-1 genomic clone demonstrated the localization of the gene near the kinetochore in chromosome 9. Thus, we confirmed that the pierisin-1 gene is located in the genome of P. rapae.

Mucosal remodeling in long-standing ulcerative colitis with colorectal neoplasia: significant alterations of NCAM+ or alpha-SMA+ subepithelial myofibroblasts and interstitial cells.

Evidence has been provided in ulcerative colitis (UC) that early genomic instability of both epithelial and stromal cells is important for colorectal tumorigenesis, as well as remodeling and morphological alterations of mucosal crypts. To clarify roles of stromal cells in tumor development in UC, the present study focused on heterogeneous phenotypes of subepithelial myofibroblasts and interstitial cells, in association with mucosal remodeling. To clarify the relationship of alterations to tumorigenesis, mucosa of resected rectae from patients with UC (n= 49) and sporadic cancer (n= 10) were analyzed on immunohistochemistry and also on immunoelectron microscopy. Heterogeneous phenotypes of neural cell adhesion molecule (NCAM)+ and/or alpha-smooth muscle actin (alpha-SMA)+ subepithelial myofibroblasts and interstitial cells were demonstrated, corresponding to colonic stellate cells. Decrease of NCAM+ subepithelial myofibroblasts and interstitial cells, and increase of alpha-SMA+ interstitial cells were significant in UC with neoplasia as compared to without neoplasia. alpha-SMA+ muscularis mucosae was significantly more thickened in tumor cases. Deposits of Masson's trichrome+ and type III and I collagen in the muscularis mucosae and lamina propria appeared to increase in relation to the numbers of alpha-SMA+ interstitial cells. Mucosal remodeling with alterations of NCAM+ or alpha-SMA+ subepithelial and interstitial cells may play a critical role in UC-associated tumorigenesis.

Effects of pre-overwintering conditions on eupyrene and apyrene spermatogenesis after overwintering in Polygonia c-aureum (Lepidoptera: Nymphalidae).

Sperm polymorphism is widely known in invertebrates. In insects, Lepidoptera has two types of sperm: nucleated eupyrene (fertile) sperm and anucleated apyrene (unfertile) sperm. These sperm types are produced during post-embryogenesis, and eupyrene spermatogenesis precedes apyrene spermatogenesis. During overwintering, spermatogenesis stops and a portion of undifferentiated-stage spermatocytes degenerate. After overwintering, spermatogenesis restarts with unaffected spermatogonia. However, how new spermatozoa arise in the adult testes after overwintering is not known in Lepidoptera. In this study, we investigated the spermatogenesis events in the nymphalid butterfly Polygonia c-aureum after overwintering under various environmental conditions. Our results indicate that both eupyrene and apyrene spermatogenesis restart at any stopping stage and sperm of these types are regenerated in no particular order after adult insect overwintering. This suggests that the spermatogenesis occurring after overwintering proceeds without embryogenetic restrictions related to the developmental sequence.

Potential Host Range of the Larval Endoparasitoid Cotesia vestalis (=plutellae) (Hymenoptera: Braconidae).

Many parasitoid wasps are highly specialized in nature, attacking only one or a few species of hosts. Host range is often determined by a range of biological and ecological characteristics of the host including diet, growth potential, immunity, and phylogeny. The solitary koinobiont endoparasitoid wasp, Cotesia vestalis, mainly parasitizes diamondback moth (DBM) larvae in the field, although it has been reported that to possess a relatively wide lepidopteran host range. To better understand the biology of C vestalis as a potential biological control of hosts other than the DBM, it is necessary to determine suitability for potential hosts. In this study, the potential host range of the wasp and its developmental capacity in each host larva were examined under laboratory conditions using 27 lepidopteran species from 10 families. The wasp was able to parasitize 15 of the 27 species successfully. Some host species were not able to exclude C vestalis via their internal physiological defenses. When parasitization was unsuccessful, most hosts killed the parasitoid at the egg stage or early first-instar stage using encapsulation, but some host species disturbed the development of the parasitoid at various stages. No phylogenetic relationships were found among suitable and unsuitable hosts, revealing that host range in some endoparasitoids is not constrained by relatedness among hosts based on immunity.

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

ESTABLISHMENT AND SOME CHARACTERISTICS OF A CONTINUOUS CELL LINE DERIVED FROM FAT BODYS OF THE CABBAGE ARMYWORM (LEPIDOPTERA, NOCTUIDAE).

Fat bodies from fully grown larvae of the cabbage armyworm, Mamestra brassicae, were cultivated in a synthetic medium, MGM-431. Small free cells migrated out and multiplied rapidly. The first cell passage was done after culture for 26 days, and 100 passages were performed in the following 9 months. The established cell line was designated as NIAS-MaBr-85. The cell population is morphologically heterogeneous, but most of the cells are hexaploid with 180 microchromosomes. The cells could be stored for 3 months at 5°C, or for longer when frozen at -80°C in medium containing 10% glycerol. Qualitatively the cell line requires few amino acids: only cystine, histidine, isoleucine, methionine and threonine are essential as determined by omission of individual amino acids. The cells are susceptible to Chilo iridescent virus and Autographa californica nuclear polyhedrosis virus. This cell line can grow in serum-free M-M medium.

PRIMARY CULTURES OF THE CELLS FROM OVARIES OF THE CABBAGE ARMYWORM, MAMESTRA BRASSICAE L. (Lepidoptera: Noctuidae).

Ovaries from post-diapause pupae of the cabbage armyworm, Mamestra brassicae, were cultured. Three types of cells migrated from the explants. Epithelial-like cells formed confluent monolayers, and fibroblast-like cells formed cellular networks. Isolated cells were distributed around the explants. Epithelial-like cells and isolated cells were common, and fibroblast-like cells were rather rare. The migrated cells survived for more than 6 months, although mitoses were rarely observed.

ESTABLISHMENT AND CHARACTERIZATION OF CONTINUOUS CELL LINES FROM PUPAL OVARIES OF THE CABBAGE ARMYWORM, MAMESTRA BRASSICAE (LEPIDOPTERA, NOCTUIDAE).

Continuous cell lines (NIAS-Mb-19, NIAS-Mb-25 and NIAS-Mb-32) were established from the ovaries of pharate adults of the cabbage armyworm, Mamestra brassicae. One-and-a-half year elapsed before the cells multiplied steadily. The continuous cell lines consisted of a heterogeneous cell population, consisting of glass-attached flat cells and spherical free cells. The former varied considerably in shape and size. Most of the cells had finely branched cytoplasmic processes. Morphologically these three lines were not distinguishable from each other. In all the cell lines, diploid cells were predominant. The adhesiveness of cells to glass differed somewhat among the different lines. The patterns of amino acid utilization of these cell lines were characterized by marked consumption of aspartic acid, cystine, glutamine, histidine, isoleucine, leucine, methionine, tryptophan, tyrosine and valine, and by production of α-alanine and proline. These cell lines were susceptible to Chilo iridescent virus, and the infected cultures maintained a carrier state for many passages. The cell lines could be preserved for a long period by freezing or for a short period by placing them in a refrigerator.

Continuous Cell Lines from Larval Hemocytes of the Cabbage Armyworm, Mamestra brassiae: (continuous cell line/hemocytes/Mamestra brassicae).

Two continuous cell lines, NIAS-MaBr-92 and NIAS-MaBr-93, were obtained from larval hemocytes of the cabbage armyworm, Mamestra brassicae. The cells grew in suspended state. Spherical cells were predominant, although there were spindle shaped and irregular shaped cells. The chromosome numbers of the cell lines varied very much with the mode of 100 of 120. The population doubling times of these cell lines were about 36hr. The cells could grow in media free of serum and lacking sterols, fatty acids and protein. The cell lines consumed ammonia and produced glutamine when cultured in MM medium. The cells could be stored at -100°C for more than two year, and at 5°C for two months. The cells were sensitive to Autographa californica nuclear polyhedrosis virus and Chilo iridescent virus.

ESTABLISHMENT OF A CELL LINE FROM EMBRYONIC TISSUES OF THE FLESHFLY, SARCOPHAGA PEREGRINA (INSECTA: DIPTERA).

A continuous cell line was established from embryonic tissues of the fleshfly, Sarcophaga peregrine, and was designated as NIH-SaPe-4. The primary culture was initiated in October, 1977, and the cell line was passed 68 times during the following year. The cells were heterogeneous in morphology. Most cells were diploid and their chromosomes consisted of 4 metacentric, 6 sub-metacentric and 2 micro chromosomes. The population doubling time of the cell line was about 30 hr. The cells grew faster in Mitsuhashi-Maramorosch's medium than in Schneider's medium. The cells were either stored in the usual medium at 5°C for about 3 months, or in a medium containing 10% glycerol at -80°C for a longer period. Cell growth was suppressed by 20-hydroxy-ecdysone when at a greater concentration than 0.01 µg/ml, whereas insulin showed no effects on cell growth at a strength of 0.4 and 0.04 IU/ml.

Spraying of phenol red dye as a screening test for Helicobacter pylori infection in surgically resected stomach specimens.

BACKGROUND: Endoscopic spraying of phenol red dye and urea (phenol red test) has recently been used to assess the distribution of Helicobacter pylori in the gastric mucosa. We examined whether the phenol red test could be used to detect H. pylori in surgically resected stomachs. METHODS: A total of 82 surgically resected stomachs, obtained from 82 patients (mean age, 60.1 years; range, 33-84 years) with early gastric carcinomas were examined. Phenol red solution and urea were sprayed over the entire mucosa of each resected stomach. A color change from yellow to red was considered as a positive reaction for H. pylori. Gastric mucosal specimens taken from positively stained and negatively stained areas on the phenol red test were then examined immunohistochemically to determine the degree of H. pylori colonization. RESULTS: Diffusely positive reactions were seen in 16 resected stomachs (19%), and regionally positive reactions were seen in 36 (44%). The other 30 stomachs (37%) showed no color change (negative reaction). H. pylori was detected immunohistochemically significantly more frequently in positively stained than in negatively stained areas ( P << 0.0001). Specificity, sensitivity, and predictive values for positive and negative results of the phenol red test, determined on the basis of H. pylori immunostaining, were 100%, 74.3%, 100%, and 72.7%, respectively. CONCLUSIONS: The phenol red test is a specific, relatively sensitive, rapid, easy-to-use, and repeatable method that can be used to diagnose H. pylori infection in surgically resected material. It enables pathologists as well as gastroenterologists with no microbiological expertise to easily diagnose H. pylori infection.

A continuous cell line from the cupreous chafer, Anomala cuprea Hope (Insecta, Coleoptera, Scarabaeidae).

A continuous cell line was obtained from the culture of embryonic cells of the cupreous chafer, Anomala cuprea Hope. The cells showed substrate-dependent growth and formed loose networks. Population doubling time was about 4.5 d. The mode of chromosome number was about 32 (4n). The cell line was designated FRI-AnCu-35.

Continuous cell lines from the common white, Pieris rapae crucivora Boisduval.

Three continuous cell lines, NIAS-PRC-819A, NIAS-PRC-819B, and NIAS-PRC-819C, were established from the pupal ovaries of the common white, Pieris rapae crucivora Boisduval (Insecta, Lepidoptera, Pieridae). The primary culture was initiated as explant cultures with ovariole fragments in MGM-464 medium supplemented with 20% fetal bovine serum at 25 degrees C. About 6 mo after the culture was set up, the first subculture was prepared. Thereafter, cells were subcultured with decreasing passage intervals, resulting in a cell population that multiplied continuously. The karyotypes of these cell lines were similar to each other, and the majority of the cells showed about 100 microchromosomes. The population-doubling times of these cell lines were 3 to 7 d. The cell lines were susceptible to a microsporidia, Nosema bombycis. Immunodiffusion experiments proved that these cell lines derived from the common white and not from other cell lines by contamination.

Differences in clinicopathological findings, cell kinetics and p53 expression between early gastric cancers with and without Helicobacter pylori infection.

BACKGROUND/AIMS: Helicobacter pylori (Hp) infection has been suggested to be a risk factor for gastric carcinogenesis. The aims of this study were to clarify differences in clinicopathological features, tumor cell kinetics and p53 expression between early gastric cancers developing within and at a distance from Hp actively infected mucosa and those away from the infected area. METHODOLOGY: A total of 91 consecutive patients who were diagnosed as having early gastric carcinoma were enrolled in this study. Phenol red solution and urea were sprayed over the surgically resected stomachs (the PR test). Tumors included in and away from positive (red color) areas on PR testing were considered as infected and non-infected cases, respectively, and compared for Ki67 (proliferative activity), ssDNA (apoptotic activity) and p53 immunoreactivity. RESULTS: The average age of the infected cases (n=46) was 9 years younger than that for their non-infected counterparts (n=45; P<0.0001). Depressed macroscopic and diffuse histologic types were more prevalent in the infected cases (P<0.05 and P<0.001, respectively). In neither intestinal nor diffuse histologic types were any significant differences in Ki67, ssDNA or p53 immunoreactivity apparent between the infected and non-infected cases. CONCLUSIONS: Cases of early gastric cancer developing within and at a distance from Hp infected mucosa are clinicopathologically different although the presence of bacteria in the surrounding mucosa does not appear to affect tumor cell kinetics or p53 expression.

Significant correlation of morphological remodeling in ulcerative colitis with disease duration and between elevated p53 and p21 expression in rectal mucosa and neoplastic development.

Although a chronic inflammation-carcinoma sequence has been proposed in cases of longstanding ulcerative colitis (UC), the relationship of morphological alteration or remodeling of regenerated mucosa to carcinoma development is yet to be clarified. Therefore, mucosae of 49 resected rectae from individuals with UC were histologically and quantitatively analyzed, with regard to thickness and morphological parameters of crypts, in relation to the disease duration, clinical disease activity and neoplastic development. An immunohistochemical examination of Ki-67, p53, p21(WAF1) and ssDNA labeling was also included. Significant correlations of number, height, angle, fusion and Paneth cell metaplasia of crypts, as well as thickness of the muscularis mucosae, were revealed with disease duration, as confirmed by three-dimensional reconstructed image analysis. p53 and p21(WAF1)-positive cells increased with disease duration and were significantly more frequent in cases with neoplasia, suggesting more DNA damage. However, this was not the case for ssDNA labeling, assessed as an indicator of apoptosis. In general, histological changes and p53, p21(WAF1) and Ki-67 labeling were correlated. In conclusion, histological parameters for mucosal remodeling correlate well with UC duration, indicating accumulation of structural alterations. Accumulated damage to DNA, reflected by increased p53 and p21(WAF1) labeling indices, might be involved in cancer development, as well as longstanding inflammation.