Sperm storage and motility in the ovary of the marine sculpin Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association.

Elkhorn sculpin, Alcichthys alcicornis, is a marine teleost with a unique reproductive mode called "internal gametic association," in which sperm introduced into the ovary by copulation enter the micropylar canal of ovulated eggs in the ovarian cavity, but actual sperm-egg fusion does not occur until the eggs have been released into sea water. It is also known that this fish is a multiple spawner, which spawns at intervals of a few days for one month, and the sperm introduced into the ovary at the beginning of the spawning season retain their fertilizability for the entire period. To clarify how the fertilizability of sperm is maintained internally, the ultrastructure of sperm, the morphological characteristics related to sperm storage in the ovary, and the characteristics of sperm motility were investigated. Mature sperm generally have the normal form of teleost sperm, devoid of acrosomal structures. However, it was found that the midpiece is comparatively elongated and has a compact aggregation of many small-size mitochondria. The intraovarian sperm remained floating in the ovarian fluid throughout the spawning season. The sperm showed high motility in isotonic and weak alkaline solution, containing sodium ions, which was similar to the ovarian fluid of this fish. Sperm continued to move in artificial ovarian fluid for 7-14 days. Considering these results together, it is thought that the intraovarian sperm move throughout the spawning season due to the plentiful energy generated by the many mitochondria.     

Application of hypothermia to autologous stem cell purging

Autologous stem cell transplantation is used widely after high-dose chemotherapy for treating hematological and other malignancies. Bone marrow harvested for autologous bone marrow transplantation may contain residual malignant cells even when the cancer is judged to be in remission. Attempts to purge marrow of its putative residual malignant cells may delay hemopoietic reconstitution and are of uncertain efficacy. In this report, we demonstrate the possibility of applying hypothermia to autologous stem cell purging. Using clonogenic assay, we compared the surviving fraction of human leukemia (HL60, K562) and human small cell lung cancer (H69) cell lines with that of normal human bone marrow CFU-GM and BFU-E cells after incubation at 4 +/- 0.1 degrees C for 24 and 48 h. Hypothermia decreased the surviving fraction of HL60, H69, and K562 cells. In contrast, the surviving fractions of stem cells were not affected by the temperature shift. The surviving fraction of HL60 cells at 4 degrees C cooling was significantly lower than that at 22 degrees C cooling. These findings suggest that in vitro hypothermia may selectively purge residual malignant cells in stored remission bone marrow and may be applicable before autologous bone marrow transplantation. In addition, the method is very simple and cost effective.     

Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.     

Serum 17,20 beta-dihydroxy-4-pregnen-3-one Levels in pregnant and non-pregnant female rockfish, Sebastes schlegeli, viviparous teleost, and its production by post-ovulatory follicles

Changes in serum 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DP) levels around the gestation time of normal pregnant and experimentally non-pregnant females were investigated in the black rockfish, Sebastes schlegeli, a viviparous teleost. The serum 17,20 beta-DP in both females showed similar changes and levels, increasing from the early to late gestation periods and declining just before parturition in pregnant females and egg-release in non-pregnant females, respectively. These results suggest that the maintenance of high serum levels of 17,20 beta-DP after oocyte maturation is not correlated with gestation or parturition, but occurs spontaneously in this species. The decline of 17,20 beta-DP levels prior to egg-release in non-pregnant females tended to occur one week earlier than those prior to parturition in pregnant females, suggesting that both a decline in 17,20 beta-DP levels in mothers and some response from embryos are needed for a smooth parturition. The post-ovulatory follicles were maintained throughout the gestation period and produced a considerable amount of 17,20 beta-DP in vitro (3.44-6.96 pg/ml/mg tissue), but little estradiol-17beta (0.92-1.66 pg/ml/mg tissue). The production of 17,20 beta-DP tended to be enhanced by the addition of a precursor steroid, pregnenolone, in the pre-, early and mid-gestation periods. These results strongly suggest that the follicle cells in black rockfish have the ability to synthesize 17,20 beta-DP during the post-ovulatory period, and high serum 17,20 beta-DP during gestation is supplied by the post-ovulatory follicles, which in Sebastes are considered to be functionally homologous to the mammalian corpus luteum.     

OBPC Symposium: Maize 2004 &; beyond—Recent advances in chloroplast genetic engineering

Summary The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrols) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.     

Environmental regulation of annual reproductive cycle in the mosquitofish, Gambusia affinis

To clarify the environmental factors regulating the annual reproductive cycle of the female mosquitofish, Gambusia affinis, a viviparous teleost, histological changes of the ovary in natural population, and laboratory experiments were examined. The results, extending over two years, suggested that ovarian recrudescence is initiated by the rise in temperature during spring and that ovarian regression is caused by the shorter daylength during late summer. The first rearing experiments using four photoperiod-temperature groups to investigate the factors triggering the onset of reproduction revealed that females with regressing ovaries began reproduction with the rise of temperature regardless of the photoperiod during spring. The results of the second experiment using three different temperature groups indicated that vitellogenesis occurred at over 14 degrees C and pregnancy at over 18 degrees C. The third experiment with four photoperiod-temperature groups was arranged to investigate the factors in the cessation of reproduction. Sexually active females ceased vitellogenesis of the next clutch of oocytes due to the shorter daylength regardless of temperatures during late summer; however, temperature seemed to influence the rate of embryo development. The critical photoperiod is estimated at about 12.5 hr. In nature, it is supposed that vitellogenesis starts when the temperature rises to about 14 degrees C, and final maturation of oocytes occurs when the temperature reaches about 18 degrees C during spring. Then, vitellogenesis of the next clutch of oocytes ceases when the daylength becomes shorter than 12.5 hr during late summer; the last gestation proceeds at a rate dependent on the temperature, and finally reproduction ends by the last parturition. J. Exp. Zool. 286:204-211, 2000.     

Incorporation and utilization of multiple forms of vitellogenin and their derivative yolk proteins during vitellogenesis and embryonic development in the mosquitofish, Gambusia affinis

We previously demonstrated the presence of three forms of vitellogenin (Vg), two 600 kDa Vgs (600Vg; VgA and VgB) and a 400 kDa Vg (400Vg; phosvitinless Vg) in plasma from maturing female viviparous mosquitofish, Gambusia affinis. For further quantitative elucidation of the accumulation and utilization of the multiple Vg-derived yolk proteins, two sandwich enzyme-linked immunosorbent assays (ELISA) were developed using antisera against 600Vgs and a 400 kDa yolk protein (400Yp; derived from 400Vg), respectively. Contents of 560 kDa yolk protein (560Yp; lipovitellins derived from 600Vg) and 400Yp measured by the ELISAs increased in accordance with the growth of vitellogenic oocytes, keeping their proportional ratio (mol/mol) at about 4:1. A similar ratio obtained for plasma Vgs suggests that the proportional accumulation of the multiple Vg-derived yolk proteins is regulated by the hepatic synthesis and secretion of their precursor Vgs. When egg homogenate was analyzed by gel chromatography, three peaks, consisting of 560Yp, 400Yp and 28 kDa native beta'-component, were observed. The elution profile showed no change until embryos reached the early neurula stage, however, the relative height of the 560Yp peak as compared to the 400Yp one decreased after retinal pigmentation. Results from measurements of 560Yp and 400Yp at each embryonic stage supported the occurrence of unequal utilization of the two yolk proteins. The proportional ratios (mol/mol) of 560Yp content versus 400Yp content gradually decreased from 4.1 fold in early neurula embryo to 1.4 fold in larva just before parturition. The present study thus demonstrated unequal utilization of the multiple Vg-derived yolk proteins in developing embryos of mosquitofish.     

Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane

Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.     

Phylogenetic position of an arbuscular mycorrhizal fungus,Acaulospora gerdemannii, and its synanamorphGlomus leptotichum, based upon 18S rRNA gene sequence

We examined the phylogenetic position of an arbuscular mycorrhizal fungus which produces two types of spore,Acaulospora gerdemannii andGlomus leptotichum, based upon the DNA sequence of the 18S rRNA gene. DNA was extracted separately from bothGlomus-like orAcaulospora-like spores and partial 5′-terminus segments of 18S rRNA gene were amplified by the PCR method. Several clones derived from each spore type were sequenced and compared. The sequences from both spore types agreed well, confirming that these morphologically different spores were formed by the same fungus. Nucleotide substitutions were found among several clones, suggesting polymorphism of the rRNA gene in glomalean fungi. Further phylogenetic analysis based upon the whole sequence of the 18S rRNA gene showed thatA. gerdemannii may be within the order Glomales but is far from the fungi that have been analyzed and probably should be in a new family.     

Mortality among Human Immunodeficiency Virus Type 2-Positive Villagers in Rural Guinea-Bissau Is Correlated with Viral Genotype

We present the results of a 6-year study of 131 human immunodeficiency virus (HIV) type 2 (HIV-2)-infected individuals from a rural population in Guinea-Bissau. Proviral DNA sequences 1.3 kb in length were obtained from each individual and, together with clinical data, including proviral load and CD4 and CD8 levels, were used to assess whether viral genotype influences clinical outcome. With a phylogenetic model, a correlation was found between viral genotype and mortality; this correlation was not due to confounding factors, such as age-specific viral strains or cohabitation of patients. The data provide strong evidence for the involvement of viral genetic factors in determining HIV disease progression in vivo. The pattern of association found suggests that virulence factors are multiple and scattered throughout the HIV-2 genome and can be rapidly gained or lost by the virus through a combination of mutation and recombination. These findings may lead to the identification of viral determinants of HIV disease progression.