Bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones: synthesis and mutagenic effects of halogen and hydroxyl group replacements

The versatility of 4-(hydroxymethyl)-2(5H)-furanone as a starting point for the synthesis of several bromine and mixed halogen analogues of the potent water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) has been demonstrated. However, in some preparations the yields of desired products were lower for bromine- than chlorine-substituted counterparts. A total of 12 bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones were tested repeatedly in 10 independent experiments for levels of Salmonella typhimurium (TA100) mutagenicities. The purpose of these experiments was to determine the mutagenic response to changing halogen content, type, and position as well as to learn the measure of these responses in the presence and absence of the C-5 OH group. Mutagenicities reached levels of 10(3) and 10(2) rev/nmol for all trihalo- and dihalo-4-methyl-5-hydroxy-2(5H)-furanones, respectively, notwithstanding substitutions by bromine or chlorine. Trihalides lacking the C-5 hydroxyl group possessed mutagenicities of the order of 10(2) rev/nmol, while hydroxyl group absence in the dihalides resulted in potency levels of slightly less than 10 rev/ nmol. Pairwise comparisons of compound mutagenicities showed that overall the C-5 H-by-OH replacement and, next in importance, increasing the number of C-6 halogens from one to two resulted in the greatest enhancements of mutagenicities. However, in comparing compound pairs within two different sets of four di- and trihalides, it was observed that replacement of a C-5 H by OH enhanced mutagenicity more for the dihalides than the trihalides indicating that increasing the C-6 halogen number simultaneously with replacing C-5 H by OH results in a nonlinear, additive enhancement. For fewer than half of the compound pairs compared, changing the C-6 halogen from chlorine to bromine resulted in small increases in mutagenicity, and for the remaining compound pairs, no increase could be discerned. This result points to the relative unimportance of only C-6 halogen type as a determinant of mutagenicity. Similarly, no impact on mutagenicity was observed for changing only the halogen type attached to C-3.     

Comparisons of the frequencies and molecular spectra of HPRT mutants when human cancer cells were X-irradiated during G1 or S phase

In an attempt to elucidate mechanisms underlying the variation in radiosensitivity during the cell cycle, mutations in the HPRT gene were selected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G1 or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with approximately 98% of the cells in G1 phase when they were irradiated after 3 h of incubation, and 75% in S phase when they were irradiated after 14 h of incubation. The mutant frequencies were approximately 4-fold higher (P < 0.01) when cells were irradiated in G1 phase compared with S phase, and the lowest frequency (1.5 x 10(-5) for 3 Gy during S phase) was approximately 10-fold higher than the spontaneous frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base-pair changes or small deletions less than 20 bp; class 2, which consisted of deletions greater than 20 bp but with one or more HPRT exons present; and class 3, which consisted of deletions encompassing the entire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A "hotspot" for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G1 phase, the percentages for the different classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontaneous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells were irradiated in G1 phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase from 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hypothesize that these differences occur because: (a) there is more error-free repair of double-strand breaks (DSBs) during S than G1 phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in each approximately 1-Mbp region 5' and 3' of the gene, cause a class 3 mutation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations.     

Agreement between persons with traumatic brain injury and their relatives regarding psychosocial outcome using the Community Integration Questionnaire

Cerebral cavernous angiomas are congenital malformations usually presenting with symptomatic hemorrhages or seizures. The advent of MRI has allowed to detect asymptomatic, "cryptic" brain cavernomas. They can occur in a sporadic, isolated form and in a familial form characterized by multiple lesions. Surgical treatment is recommended by most authors in symptomatic cases when the lesion is isolated and readily accessible. Treatment is more controversial when the vascular malformation is located in critical brain region. On the other hand the increased risk of rebleeding after a prior hemorrhage and the decreased effectiveness of lesionectomy in patients with a long seizure history could lead to perform an early surgical excision also for cavernous angiomas located in those areas. We report three cases of symptomatic cerebral cavernomas located in critical areas, in children. In all cases the surgical removal of the lesions was successfully performed. The two cases presented with seizures are seizure-free in spite of the interruption of the antiepileptic therapy. The patient presented with neurological deficit due to intraparenchymal hemorrhage progressively recovered his motor function. Our experience seems to confirm the value of surgical removal regardless of where the cavernoma is located.     

Mutational analysis of the BRCA1 tumor suppressor gene in endometrial carcinoma

OBJECTIVE: To investigate whether alteration of BRCA1 tumor suppressor gene occurs in sporadic endometrial carcinomas. METHODS: Genomic DNAs were prepared from 33 consecutively collected endometrial carcinoma tissues for BRCA1 mutational analysis. To screen for BRCA1 mutation, polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) technique were used with 41 overlapping PCR primer pairs for the 23 coding exons of BRCA1. Tumors that demonstrated SSCP variants were further subjected to direct DNA sequencing in the appropriate exons to identify the DNA alteration. RESULTS: In addition to detecting a previously described polymorphism in exon 11, single strand conformation polymorphism analysis of the 33 endometrial cancers identified 3 tumors with mobility shifts. Two tumors shifted in exon 3 and showed the same pattern of band shift. The other tumor shifted in exon 9. DNA sequencing revealed sequence alterations in the 3 tumors; all appeared heterozygous. In the 2 tumors shifted in exon 3, the sequence alteration caused no amino acid change and was consistent with an infrequent silent polymorphism. In the third tumor, a missense alteration at codon 191 was detected and was recognized as germline in origin. CONCLUSIONS: Because a normal allele of BRCA1 was retained in the tumor where a germline missense alteration was detected, the heterozygous DNA alteration should not be cancer predisposing in terms of the two-hit model for inactivation of the tumor suppressor gene. We conclude that mutation of BRCA1 may not be involved in the development of sporadic endometrial cancer.     

Ketorolac (Toradol) as an analgesic in swine following transluminal coronary angioplasty

The arch forms of 38 cases (53 nonextraction and 23 extraction arches) in which expansion, while maintaining arch form, was the objective of the practitioner, were analyzed before treatment, after treatment, and an average of 6 to 8 years after retention. The cubic spline was used to fit a curve representing arch form. By superimposing the spline curves, changes in arch form were analyzed with the variables rebound change (RC), rebound index (RI), rebound number (RN), and stability number (SN). Traditional linear intraarch dimensions were also analyzed. Analysis of variance was used to determine differences between the maxillary and mandibular arches and between the extraction and nonextraction cases. Pearson correlation coefficients between spline variables and arch width variables were also computed. There was significantly more expansion in the maxillary arch than the mandibular arch during treatment, irrespective of extraction or nonextraction strategies. In the nonextraction cases, a greater amount of net expansion was achieved for all dimensions for the maxillary arch as compared with the mandibular arch. Overall, a relatively high stability in arch form was found. The findings suggest that stability may not be related to the amount of change produced during treatment. Significant expansion can be gained throughout the premolar regions and may be expected to be stable. The order of greatest net arch width gained was for the second premolars followed by first premolars, molars, and then the canines. The intercanine widths for both arches decreased toward pretreatment values, but were more stable in the maxillary arch in nonextraction cases. The cubic spline permits measurement of change in arch form both during treatment and retention periods.     

Prospective, multicenter study of laparoscopic ventral hernioplasty. Preliminary results

BACKGROUND: A standard technique for laparoscopic ventral hernioplasty (peritoneal onlay using an expanded polytetrafluoroethylene [ePTFE] patch for hernias >/=4 cm2) is being used in a prospective, multicenter, long-term study. METHODS: Demographic, operative, and postoperative data were collected and analyzed. Follow-up clinical evaluations were conducted 7-10 days, 4 weeks, 6 months, 1 year, and then annually after surgery in all patients. RESULTS: In the first 2 years of the study, 144 patients were enrolled; nine were lost to follow-up. The mean operating time was 120 min. The mean follow-up was 222 days (range 5-731). Postoperative complications were five infections, three cases of prolonged ileus, one bowel obstruction, 23 seromas (15 resolved without intervention), and six hernia recurrences. Hospital discharge occurred a mean of 2.3 days after surgery and return to normal activity a mean of 15 days postoperatively. CONCLUSIONS: Laparoscopic prosthetic ventral hernioplasty avoids the large wound required in open repairs, with attendant complications and recurrences, and appears safe, especially if an ePTFE mesh is used. Compared with conventional open ventral hernioplasty, the laparoscopic technique may also allow shorter hospitalization and a quicker return to normal activities after surgery.     

The dense granule antigen, GRA2 of Toxoplasma gondii is a glycoprotein containing O-linked oligosaccharides

A high-performance liquid chromatographic method for the determination of DRF-2189, using troglitazone as internal standard, is described. A dichloromethane-ethyl acetate solvent mixture (6:4, v/v) was used as the extraction solvent. A Kromasil C18 column with a mobile phase consisting of 0.05 M phosphate buffer-acetonitrile-methanol (22.5:37.5:40) (pH 5.0) was used at a flow-rate of 1.0 ml/min. The eluate was monitored by using fluorescence detection with excitation and emission wavelengths at 292 nm and 325 nm, respectively. Ratio of peak area of analyte to internal standard was used for quantification of plasma samples. Using this method, the absolute recovery of DRF-2189 from rat plasma was >95% and the limit of quantitation was 50 ng/ml. The intra-day relative standard deviation (R.S.D.) ranged from 1.74 to 7.24% at 1 microg/ml and 1.86 to 3.83% at 10 microg/ml. The inter-day R.S.D.s were 8.34 and 4.91% at 1 and 10 microg/ml, respectively. The method was applied to measure plasma concentrations of DRF-2189 in pharmacokinetic studies in Wistar rats.     

Nocturnal elevation of intraocular pressure in young adults

PURPOSE: To distinguish 24-hour (circadian) and postural effects on intraocular pressure (IOP) in healthy young adults. METHODS: Thirty-three volunteers were housed in a sleep laboratory for 1 day under a strictly controlled 16-hour light and 8-hour dark environment. Sleep was encouraged in the dark period. Intraocular pressure was measured in each eye every 2 hours using a pneumatonometer. Researchers used night-vision goggles to perform IOP measurements in the dark, while the subject's light exposure was minimized. In the first group of 12 subjects, measurements were taken with subjects in the sitting position during the light-wake period and supine during the dark period. In the second group of 21 subjects, all IOP measurements were taken with the subjects supine. RESULTS: Average IOP was significantly higher in the dark period than in the light-wake period in both groups. The lowest IOP occurred in the last light-wake measurement, and the peak IOP occurred in the last dark measurement. The trough-peak difference in IOP was 8.2+/-1.4 mm Hg (mean +/- SEM) in the first group. Intraocular pressure changed sharply at the transitions between light and dark. In the second group, the trough-peak IOP difference was 3.8+/-0.9 mm Hg. Intraocular pressure changed gradually throughout the 24-hour period. In comparison with the sitting IOP in the first group, the supine IOP in the second group was significantly higher during the light-wake period. CONCLUSIONS: Circadian rhythms of IOP were shown in young adults, with the peaks occurring in the late dark period. A nocturnal IOP elevation can appear independent of body position change, but change of posture from upright to recumbent may contribute to the relative nocturnal IOP elevation.     

Interleukin-2 triggers a novel phosphatidylinositol 3-kinase-dependent MEK activation pathway

Phosphatidylinositol 3-kinase (PI3-K) has been implicated as a signal-transducing component in interleukin-2 (IL-2)-induced mitogenesis. However, the function of this lipid kinase in regulating IL-2-triggered downstream events has remained obscure. Using the potent and specific PI3-K inhibitor, wortmannin, we assessed the role of PI3-K in IL-2-mediated signaling and proliferation in the murine T-cell line CTLL-2. Addition of the drug to exponentially growing cells resulted in an accumulation of cells in the G0/G1 phase of the cell cycle. Furthermore, wortmannin also partially suppressed IL-2-induced S-phase entry in G1-synchronized cells. Analysis of IL-2-triggered signaling pathways revealed that wortmannin pretreatment resulted in complete inhibition of IL-2-provoked p70 S6 kinase activation and also attenuated IL-2-induced MAP kinase activation at drug concentrations identical to those required for inhibition of PI3-K catalytic activity. Wortmannin also diminished the IL-2-triggered activation of the MAP kinase activator, MEK, but did not inhibit activation of Raf, the canonical upstream activator of MEK. These results suggest that a novel wortmannin-sensitive activation pathway regulates MEK and MAP kinase in IL-2-stimulated T lymphocytes.