Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes

The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.     

Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface.

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.     

First cutaneous branch of the internal pudendal artery: an anatomical basis for the so-called gluteal fold flap

We investigated the cutaneous blood supply in the gluteal and perineal regions of 35 donated cadavers to provide an anatomical basis for reliable vulvo-vaginal reconstruction using a skin flap such as the so-called gluteal fold flap. The cutaneous areas along the gluteal cleft and sulcus were likely to be supplied by 3 routes: 1) the internal pudendal artery (IPA), especially its first cutaneous branch; 2) perforators running through the gluteus maximus muscle and arising from the inferior gluteal artery (IGA); and 3) a non-perforator running around and inferior to the ischial tuberosity and originating from the IGA. Route 1 supplied the skin along the gluteal cleft, route 2 the gluteal fold (i.e., a bulky skin fold along the upper edge of the gluteal sulcus), and route 3, just along the gluteal sulcus. In those 3 routes, we noted the consistent morphology of the thick and long, first cutaneous branch of the IPA. The first arterial branch, 1.5 mm in diameter at its origin on average (ranging from 0.7-2.6 mm), usually originated from the IPA under the cover of or at the inferomedial or distal side of the sacrotuberous ligament (almost always less than 20 mm from the inferomedial margin of the ligament). The branch ran superomedially toward the coccyx or ran medially in the ischiorectal fat. It accompanied the vein and nerve at its distal (peripheral) course although the nerve often ran independently at its proxomal course near the ligament. Therefore, the first branch of the IPA seems to provide a reliable pedicle using the skin along the gluteal cleft whether the incision for approach is conducted along the gluteal sulcus or not. However, if the gluteus maximus muscle extended much inferomedially, the pedicle would be very short. In this case, preparation of the pedicle seems to be necessary along the arterial course under the cover of the muscle.     

Inhibitory effect of estradiol-17 beta and progesterone on bactericidal activity in uteri of rabbits infected with Escherichia coli.

The influence of ovarian hormones at different estrous stages on the bactericidal activity of the uterus in rabbits was investigated. When Escherichia coli cells were inoculated in ligated uteri, the survival period of the bacteria in the uterus at the luteal phase was clearly longer than that at the follicular phase. At the luteal phase, high levels of plasma estradiol-17 beta and progesterone were detected. A luteolytic treatment with prostaglandin F2 alpha and human chorionic gonadotropin at the luteal phase lowered plasma progesterone levels and prompted bacterial clearance from the uterus. In ovariectomized rabbits, E. coli from the uterine exudates was not detected 6 days after the inoculation in both the nontreated and estradiol-17 beta-treated animals. In the progesterone-treated rabbits, the survival period of E. coli was longer than that in the nontreated and estradiol-17 beta-treated animals. When estradiol-17 beta and progesterone at the ratio of 1:100 were administered concurrently, E. coli survived for the longest period in the rabbits treated with various doses of different hormones. Formalin-killed E. coli cells were inoculated into the uterine lumen, and 4 h later the proportion of heterophils phagocytizing the bacteria dropped in the progesterone-treated rabbits and in the estradiol-17 beta- and progesterone-treated rabbits, but there was no significant difference in heterophil numbers among the rabbits treated with different hormones. The present results suggest that progesterone inhibits the bactericidal activity of the uterus and that estrogen concurrently secreted at the luteal phase promotes the inhibitory action of progesterone, although estrogen alone hardly affects the uterine defense. In addition, the lowering of the bactericidal activity of the uterus at the luteal phase may be attributable to lower activity of phagocytosis by heterophils infiltrated into the uterine lumen.     

Increased peripheral blood Ia positive T cells and their effect on autologous mixed lymphocyte reaction in chronic active liver disease.

We measured Ia antigen bearing peripheral blood T cells, as an index of immunological stimulation, of patients with chronic active liver diseases (CALD) by the rosette assay method. We also examined the role of Ia antigen which represents the products of the genes of the major histocompatibility complex on the autologous mixed lymphocyte reaction (AMLR) since this reaction may reflect self regulation of immune responses. The percentages of Ia positive T cells of 29 patients with CALD (17.1 +/- 4.3%, P less than 0.001) and of 12 patients with other liver diseases (12.9 +/- 2.4%, P less than 0.05) were increased when compared with that of normal individuals (10.7 +/- 2.0%). However, levels of Ia positive T cells activated by phytohaemagglutinin-P in patients with CALD and other liver diseases did not differ from normal subjects. Ia positive cells in OKT8 positive cells were markedly elevated (P less than 0.001), whereas those in OKT4 positive cells were decreased (P less than 0.01) in CALD. The impaired values for the AMLR correlated inversely (P less than 0.01) with the increased percentages of Ia positive T cells in patients with CALD. Further analysis showed that there was no suppression of the proliferation of Ia and OKT4 positive cells by Ia and OKT8 positive cells although the culture of increasing numbers of Ia and OKT8 positive cells and decreasing numbers of Ia and OKT4 positive cells gave a lesser AMLR value. These data suggest that the increase in Ia positive T cells and the alteration of Ia positive cells in the T cell subsets reflect an activation of immune system and provide further evidence in favour of an abnormality of the immunoregulatory system in CALD.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

P16-immunostaining pattern as a predictive marker of lymph node metastasis and recurrence in early uterine cervical cancer.

OBJECTIVES: We investigated the relationship between P16-immunostaining patterns and clinicopathological factors in early uterine cervix cancers and assessed whether P16-immunostaining patterns predict the prognosis of the patients with early uterine cervix cancers. METHODS: Twenty-nine early squamous cell carcinoma (SCC) specimens of the uterus were examined using immunohistochemistry for P16 expression. The P16-immunostaining pattern was classified into two groups: the homogeneous type and the heterogeneous type. P16-immunostaining patterns were evaluated in different parts of the carcinoma in situ (CIS): the center of the tumor and the front interface of the infiltrating tumor. RESULTS: All specimens were of the homogeneous type in CIS. The P16-immunostaining pattern was significantly of the heterogeneous type in the front interface of the infiltrating tumor with lymphatic invasion, vascular invasion, lymph node metastasis, and recurrence. Regarding the P16-immunostaining patterns in the front interface of the infiltrating tumor, the patients with the heterogeneous type showed a significantly worse prognosis than the patients with the homogeneous type. CONCLUSIONS: The prognosis of patients with early uterine cervical SCC may be predicted by evaluating the P16-immunostaining pattern in the front interface of the infiltrating tumor.