Intermediate-risk grouping of cervical cancer patients treated with radical hysterectomy: a Korean Gynecologic Oncology Group study.

BACKGROUND: In this study, we sought to identify a criterion for the intermediate-risk grouping of patients with cervical cancer who exhibit any intermediate-risk factor after radical hysterectomy. METHODS: In total, 2158 patients with pathologically proven stage IB-IIA cervical cancer with any intermediate-risk factor after radical hysterectomy were randomly assigned to two groups, a development group and a validation group, at a ratio of 3 : 1 (1620 patients:538 patients). To predict recurrence, multivariate models were developed using the development group. The ability of the models to discriminate between groups was validated using the log-rank test and receiver operating characteristic (ROC) analysis. RESULTS: Four factors (histology, tumour size, deep stromal invasion (DSI), and lymphovascular space involvement (LVSI)) were significantly associated with disease recurrence and included in the models. Among the nine possible combinations of the four variables, models consisting of any two of the four intermediate-risk factors (tumour size ≥3 cm, DSI of the outer third of the cervix, LVSI, and adenocarcinoma or adenosquamous carcinoma histology) demonstrated the best performance for predicting recurrence. CONCLUSION: This study identified a 'four-factor model' in which the presence of any two factors may be useful for predicting recurrence in patients with cervical cancer treated with radical hysterectomy.

Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast.

The present studies were carried out to investigate the effects of intensity of dc pulse, number of dc pulse and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. In experiment 1, different fusion/activation intensity (two dc pulses of 0.4, 0.8, 1.2, 1.6 and 2.0 kV/cm for 30 micros, respectively) was carried out to investigate development of embryos. In experiment 2, the reconstructed oocytes were fused and activated with one, two or three dc pulses of 1.2 kV/cm for 30 micros. In experiment 3, reconstructed oocytes were equilibrated in TCM-199 medium for 0-6 h, respectively, and fused/activated with one dc pulse of 1.2 kV/cm for 30 micros. The reconstructed embryos were cultured in PZM-3 medium containing 0.3% BSA. When oocytes were fused with donor cell by two dc pulses of 0.4 kV/cm for 30 micros, the rates of cleavage and blastocyst formation were significantly lower (32.9% and 2.5%) than those of fused by 0.8 kV/cm (59.0% and 17.4%) or 1.2 kV/cm (63.3% and 18.4%), respectively. One dc pulse of 1.2 kV/cm for 30 micros was enough to fuse and activate embryos to develop to blastocyst (24.8%). Equilibration for 2-3 h in TCM-199 before fusion/activation was beneficial for improving the developmental ability of embryos produced by nuclear transfer (25.6-23.3% at blastocysts).

Pad - a new self-collection device for human papillomavirus.

This study was designed to determine the accuracy and agreement of a self-collection method using pad for human papillomavirus (HPV) DNA. One hundred and thirty-four patients at university hospitals voluntarily participated in the accuracy study, and 314 volunteers participated in the agreement study at local clinics. DNA was extracted and amplified using HPV L1 consensus primers designed for the direct sequencing. In the accuracy study, all samples were probed via histological examinations. With regard to the detection of squamous intraepithelial lesion (SIL), self-collection pad sampling displays sensitivity, of 76.9%, and specificity, of 93.3%. Three hundred and fourteen self-collection pad samples and the concurrent physicians' samples showed a 97.8% agreement, with a Kappa value of 0.9200. A new self-collection pad for the detection of HPV DNA appears to constitute an easy, rapid, and convenient alternative method for the cervical cancer screening of many women with the virtue of being incredible readily accessible.

Sleep-related nocturnal erections and erections during midazolam-induced sedation in healthy young men.

This study was performed to evaluate the characteristics of penile erection during midazolam-induced sedation after nocturnal sleep deprivation (NSD) and to determine the effect of NSD on erectile episodes in healthy, sexually functional young men. This procedure might possibly prove to be a brief office-based method of assessing whether erectile dysfunction is psychogenic or biogenic. Nineteen volunteers between the ages of 20 and 29 years participated in this study. We measured the morning penile erection after midazolam (3-5 mg) administration intravenously and all subjects completed 42 tests. Of 42 test, 28 tests revealed erectile episodes, whereas no erectile episodes were observed in 14 tests. Nocturnal sleep deprivation rate was significantly higher in tests with erectile episodes than in tests without erectile episode (P=0.030). Test order or duration of test was not different between two test results. Number of erectile episodes (r=0.374, P=0.015), tip radial rigidity (r=0.412, P=0.007), base radial rigidity (r=0.366, P=0.017) and tip tumescence (r=0.447, P=0.003) correlated with the degree of NSD. When we determined whether NSD was discriminative with regard to erectile episodes, the area under the receiver operating characteristic curve was calculated at 0.705 (95% confidence interval, 0.527-0.883; P=0.032) for the possibility of erectile episodes. Nocturnal sleep deprivation might recover the inhibited rapid eye movement sleep during midazolam-induced sedation. Our findings suggest that erection monitoring during midazolam-induced sedation after NSD may be convenient. However, validation of midazolam-induced morning penile tumescence monitoring with a large population is mandatory.

Rat and human natural killers exhibit contrasting immunoglobulin G subclass specificities in antibody-dependent cellular cytotoxicity reflecting differences in their Fc receptors (Fc gamma R).

Rat and human natural killers (rtNK and huNK, respectively) were compared in quantitative antibody-dependent cellular cytotoxicity (ADCC) assays for their capacity to recognize mouse and rat IgG monoclonal antibodies (MAb) of different subclasses. NK from these two species exhibit considerably different patterns of IgG subclass recognition as determined by the relative antibody concentrations required for comparable levels of target cells lysis. ADCC assays with a panel of 16 MAb revealed that the efficiency of rtNK-mediated target lysis diminished according to IgG subclass in the following order: molgG1 greater than rtlgG2a greater than molgG2b approximately molgG2a greater than rtlgG2b greater than molgG3. By comparison, huNK recognized the same antibodies with nearly the opposite order of efficiency: rtlgG2b much greater than molgG2a greater than molgG3 greater than molgG2b much greater than rtlgG2a approximately molgG1. Only molgG2a antibodies were equally potent with rtNK and huNK. The contrasting difference in IgG subclass recognition by rat and human NK reflects the comparatively low protein sequence homology between their respective IgG Fc receptors (Fc gamma R).

The Bradyrhizobium japonicum hsfA gene exhibits a unique developmental expression pattern in cowpea nodules.

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the beta-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5' of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5' of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.

Stimulation of melanogenesis by glycyrrhizin in B16 melanoma cells.

Glycyrrhizin (GR), triterpenoid saponin composed of one glycyrrhetinic acid (GA) and two glucuronic acids, is a main constituent of the hydrophilic fraction of licorice (Glycyrrhiza glabra) extracts and is known to have a wide range of pharmacological actions. In this study, we investigated the mechanism of GR effect on melanogenesis in B16 murine melanoma cells. The cellular levels of tyrosinase mRNA, protein, enzyme activities and melanin contents were increased by GR in a dose dependent manner. Expression of tyrosinase-related protein-2 (TRP-2) mRNA was also increased by GR, however, no significant change was observed on TRP-1. No cytotoxicity was observed at the effective concentration range of GR. GA showed no effect on melanogenesis at the equivalent nontoxic concentrations, indicating that glycoside structure is important in the stimulatory effect of GR on melanogenesis. These results indicate that GR-induced stimulation of melanogenesis is likely to occur through the transcriptional activation.

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens.

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.

Cross-talk between N-methyl-D-aspartate and adrenergic neurotransmission in the regulation of hypothalamic GnRH gene expression.

Although it has been known that activation of N-methyl-D-aspartate (NMDA) receptor effectively stimulates GnRH biosynthesis and release from the rat hypothalamus, no evidence that NMDA receptors exist in GnRH neurons is yet available. It is then presumed that the action of NMDA on GnRH neurons may be indirectly mediated through interneurons, such as catecholamines. The present study is designed to investigate whether the effect of NMDA on GnRH gene expression is mediated by adrenergic neuronal system. Adrenergic receptor antagonists were administered 30 min prior to NMDA administration to immature male rats and then animals sacrificed 60 min after NMDA administration. GnRH mRNA levels were determined by Northern blot analysis using a GnRH RNA probe. Inhibition of either alpha 1 adrenergic receptor with prazosin or beta adrenergic receptor with propranolol did not cause any change in the basal GnRH mRNA levels but reduced NMDA-induced GnRH mRNA levels. However, inhibition of alpha 2 adrenergic receptor with yohimbine increased GnRH mRNA levels but did not affect NMDA-induced GnRH mRNA levels. These findings suggest that the effect of NMDA on GnRH gene expression is mediated through adrenergic neurotransmission.

Higher incidence of p53 mutation in cervical carcinomas with intermediate-risk HPV infection.

OBJECTIVE: Inactivation of p53, either through mutation or interaction with human papillomavirus (HPV) E6 oncoprotein, is a characteristic feature of cervical carcinoma cell lines that have been previously studied. To elucidate the role of p53 in the carcinogenesis of Korean cervical carcinomas, 27 HPV-positive and 13 HPV-negative cervical carcinomas were studied in order to evaluate the status of the p53 gene. STUDY DESIGN: The HPV status was ascertained by polymerase chain reaction (PCR) amplification using consensus primers designed from the E6 and E7 open reading frames (ORFs). The p53 mutation status was analyzed by direct sequencing of the PCR product in highly conserved exons 5-8. RESULTS: There was no significant difference in the frequency of the p53 mutation between the HPV-positive and negative cases. All three mutations in the HPV-positive cases were associated with intermediate-risk viruses. The average age of the patients with the p53 mutation was 14 years older than that of patients without the p53 mutation. CONCLUSION: p53 mutations are higher in the so called intermediate-risk HPV positive than HPV 16 or 18 positive cervical carcinomas.

Non-amplification of an allele of the D8S1179 locus due to a point mutation.

During a population study of 128 Korean families (626 persons) with the AmpF/STR Profiler Plus PCR amplification system, we found an unusual homozygous genotype at the D8S1179 locus in 4 families. Therefore, a new pair of primers was designed for the D8S1179 locus from GenBank data (GenBank Accession No. G08710) to evaluate the cause. The newly designed primers amplified alleles that were not amplified with the AmpFlSTR Profiler Plus PCR amplification system. We sequenced alleles of the family members who had non-amplified alleles and we found a G-to-A transition at the position of the 147th base of the GenBank sequence.

Empty and peptide-containing conformers of class I major histocompatibility complex molecules expressed in Drosophila melanogaster cells.

Transfected Drosophila melanogaster cells can express large quantities of class I major histocompatibility complex molecules. Such molecules lack endogenous peptides because the Drosophila cells are devoid of proteins necessary for intracellular peptide loading. The empty molecules are efficiently expressed on the cell surface and can acquire extracellular peptides. The conformation and stability of empty murine class I molecules are determined by the source of beta 2-microglobulin. All beta 2-microglobulin-induced conformers of empty heavy chains seem to be unified in a common rigid conformation on peptide binding.

In vivo regulation of the assembly and intracellular transport of class I major histocompatibility complex molecules.

Using H-2Kb-transfected Balb/c 3T3 cells which generate "empty" H-2Kb molecules devoid of antigenic peptides, we show that peptide availability determines the stability of class I molecules and dictates the overall intracellular transport rate of the class I complexes. Our data also indicate that chaperonin-like proteins are involved in class I assembly. Using Drosophila cells transfected with H-2Kb and murine beta 2-microglobulin, we show that one possible candidate, calnexin, associates with class I molecules prior to peptide acquisition. These data suggest that both peptide supply and assembly proteins dictate cell surface expression of class I major histocompatibility complex molecules and ultimately influence T cell recognition. The role of beta 2-microglobulin in class I assembly is also discussed.

In vitro peptide binding to soluble empty class I major histocompatibility complex molecules isolated from transfected Drosophila melanogaster cells.

A soluble form of a mouse class I major histocompatibility antigen (H-2Kb) has been expressed in transfected Drosophila melanogaster cells. These molecules were efficiently secreted (up to 4 mg/liter) as noncovalent heterodimers and purified to homogeneity from cell supernatants. The isolated soluble Kb molecules were devoid of endogenous peptides. Using these molecules, we have characterized the Kb heavy chain-beta 2-microglobulin (beta 2m) assembly as well as peptide binding in vitro. In detergent-free solution the heavy chains readily re-assembled with beta 2m even in the absence of peptides. Kinetic analyses showed that the peptide binding is rapid and reversible and dependent on the heavy chains being assembled with beta 2m. Likewise, peptide dissociated from Kb molecules without the displacement of beta 2m. Equilibrium binding experiments using various peptides confirmed that octapeptides bind to Kb molecules with the highest affinity and form the most stable complexes. However, in contrast to earlier studies, the amino-terminal positioning of peptide to Kb molecules was more crucial than the carboxyl-terminal positioning and amidation of the peptide carboxylate did not affect the binding. Soluble Kb molecules could selectively bind allele-specific peptides among a mixture of randomly synthesized octapeptides in vitro; however, no dominant residue was observed at the carboxyl terminus of bound peptides. This suggests that the previously observed hydrophobic residues at the carboxyl terminus of peptides may reflect the specificity of enzyme(s) or protein(s) involved in peptide processing in vivo.

The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and -2 superdomain.

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2Ld molecule exhibited enhanced cross-reactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2Ld class I hybrid molecules were then used to establish the importance of the alpha-2 and -3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2Ld hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2Ld molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2Ld can affect the conformation of the alpha-1 and -2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.

Allospecific cytotoxic T lymphocytes recognize an H-2 peptide in the context of a murine major histocompatibility complex class I molecule.

We have isolated cytotoxic T lymphocytes (CTL) preferentially reactive with the alpha 1 external domain of the H-2Ld antigen by selecting for T cells capable of recognizing a variant major histocompatibility complex (MHC) class I antigen sharing alpha 1 sequences with H-2Ld. Using these CTL, we demonstrate that a synthetic alpha 1 peptide corresponding to one of the helices derived from the H-2Ld molecule can be presented by a class I restriction element to reconstitute a CTL determinant borne by intact H-2Ld. Moreover, several other H-2L-reactive CTL generated independently were also able to recognize H-2Ld either as an intact alloantigen or as a peptide in conjunction with appropriate class I restriction elements. These data demonstrate that an H-2 peptide can reconstitute a CTL target structure and suggest that some alloreactive T cells in fact might be directed against allogeneic class I peptides in the context of a class I framework.

Comparison of the synergistic effects of tamsulosin versus phentolamine on penile erection: in vitro and in vivo studies.

In vitro and in vivo studies were performed to determine the potential use of tamsulosin (TAM) versus phentolamine (PHE) for intracavernosal injection (ICI) therapy when mixed with papaverine (PAP) and/or prostagladin E1 (PGE1) or with vasoactive intestinal polypeptide (VIP) for the treatment of erectile dysfunction. We performed isometric tension studies on rabbit (n = 15), dog (n = 5), and human (n = 10) cavernous smooth muscle strips with TAM, PAP, PHE, VIP, PGE1, and the combinations of PAP and PHE; PAP and TAM; VIP and PHE; VIP and TAM; PAP, PGE1 and PHE; and PAP, PGE1 and TAM. TAM-containing trimix (PAP 18.75 mg, PGE1 6.25 micromg, and TAM 0.875 mg per ml) or PHE-containing trimix (PAP, PGE1, and PHE 0.625 mg per ml) were also injected into the cavernous bodies of ten mongrel dogs. Among the single agents, TAM and PGE1 (only in human) had the strongest effect on the relaxation of cavernous muscles in rabbit, dog, and human strips (P<0.05). Relaxation responses to 2- or 3-drug mixtures containing tamsulosin were also significantly better (P<0.05) than PHE-containing ones in rabbit, dog, and human strips. The increase in intracavernosal pressure with a TAM-containing trimix was higher than with a PHE-containing one (0.03 ml; 81.2 vs. 75.8 mm Hg, 0.04 ml; 103.2 vs. 94.3 mm Hg), although not statistically different. The drop in systemic blood pressure was lower after injection of a TAM-containing trimix than a PHE-containing one, although not statistically different. In conclusion, tamsulosin might be a more efficacious and safer agent to use for ICI therapy than phentolamine.

Tick bites in Korea.

BACKGROUND: Tick bites are dermatoses not commonly encountered in Korea. Recognizing their clinical signs as well as their histopathologic findings is important in making a diagnosis of tick-related dermatoses. The incidence and causative species are different depending on the geographic areas. The histopathologic findings of tick bites are known to be a variable depending on the species of ticks involved and the duration of their bloodsucking. METHODS: Five ticks were collected from five patients and three of them were identified as Ixodes (I.) nipponensis. RESULTS: Histopathologic findings of panniculitis were prominent in four of five cases; septal panniculitis in two cases, and lobular panniculitis in the other cases. CONCLUSIONS: Ixodes nipponensis was the most common causative species of ticks responsible for tick bites in Korea, and tick bite panniculitis must be considered in the differential diagnosis of panniculitis which is mainly composed of neutrophils.

Adenoma malignum of uterine cervix in Peutz-Jeghers syndrome: CT and US features.

We report CT and ultrasound (US) features of adenoma malignum of the uterine cervix in a patient with Peutz-Jeghers syndrome (PJS) in whom bilateral ovarian mucinous cystadenomas and sex cord tumors with annular tubules were associated. Adenoma malignum was shown as a hyperechoic mass mixed with multiple cysts on US and a low attenuated endocervical mass on CT. We think that imaging demonstration of an endocervical mass is important for the correct diagnosis of adenoma malignum in a female with PJS.

Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates.

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.