Optimization of a ß-Lactam Scaffold for Antibacterial Activity via the Inhibition of Bacterial Type I Signal Peptidase.

ß-Lactam antibiotics, one of the most important class of human therapeutics, act via the inhibition of penicillin-binding proteins (PBPs). The unparalleled success in their development has inspired efforts to develop them as inhibitors of other targets. Bacterial type I signal peptidase is evolutionarily related to the PBPs, but the stereochemistry of its substrates and its catalytic mechanism suggest that ß-lactams with the 5S stereochemistry, as opposed to the 5R stereochemistry of the traditional ß-lactams, would be required for inhibition. We report the synthesis and evaluation of a variety of 5S penem derivatives and identify several with promising activity against both a Gram-positive and a Gram-negative bacterial pathogen. To our knowledge these are the first 5S ß-lactams to possess significant antibacterial activity and the first ß-lactams imparted with antibacterial activity via optimization of the inhibition of a target other than a PBP. Along with the privileged status of their scaffold and the promise of bacterial signal peptidase I (SPase) as a target, this activity makes these compounds promising leads for development as novel therapeutics.

Unilateral Vocal Cord Paralysis following Insertion of a Supreme Laryngeal Mask in a Patient with Sjögren's Syndrome.

Since its introduction in 1988 by Dr. Archie Brain, the laryngeal mask airway (LMA) is being used with increasing frequency. Its ease of use has made it a very popular device in airway management and compared to endotracheal intubation it is less invasive. The use of LMA was on the rise, so has been the incidence of its related complications. We report severe unilateral vocal cord paralysis following the use of the supreme laryngeal mask (sLMA) in a patient with Sjögren's syndrome. In addition, we propose possible mechanisms of injury, review the existing case reports, and discuss our findings.

The efficacy of stereotactic radiotherapy for metastases from renal cell carcinoma.

Our purpose was to evaluate the efficacy of stereotactic radiotherapy (SRT) for intracranial and extracranial metastases in patients with renal cell carcinoma. The retrospective analysis of 85 patients (151 tumors) treated with SRT was performed. SRT was the sole treatment in 35% of tumors, the other 65% had received additional treatment such as surgery, palliative radiotherapy, immunotherapy or chemotherapy. In 60% and 40% of patients SRT was delivered to brain and extracranial lesions, respectively. The assessment of the efficacy of SRT was based on a radiological imaging (Computed Tomography or Magnetic Resonance Imaging) and estimation of Local Control (LC) as well as Overall Survival (OS). Single fraction was used for 104 tumors and fractionated treatment for 47 tumors. The crude LC for evaluable lesions was 81%, stratified by tumor location: brain LC=94%, extracranial tumors LC=70% (p=0.049). The median OS was 9.4 months; 1-year and 2-year OS were 40% and 29%, respectively. The additional treatment did not lead to a better local response (p=0,543), but resulted in a benefit in OS (7 vs 13 months, p=0,01). A positive relationship between the biologically effective dose (BED) and local response was noted, but the BED was influenced by a tumor volume (R=-0,38; p<0,00001). The presence of multi-organ metastases reduced the OS rate (8.7 vs 19.1 months; p=0,01). The interval between the diagnosis of the metastasis and its treatment with SRT was inversely related to OS (P=0.0001). SRT results in a good local response, which is more beneficial for brain than extracranial lesions. The local efficacy of the SRT depends on the radiation dose. Multidisciplinary treatment and earlier application of SRT improves the prognosis of patients.

Synthesis of Lepadiformine Using a Hydroamination Transform.

Dissection of lepadiformine by a double hydroamination transform affords a simple achiral amino diene. This reaction is accomplished in the forward sense by amine-directed hydroboration and an oxidative alkyl shift to nitrogen, both of which occur with high stereoselectivity to generate three stereogenic centers and the lepadiformine skeleton.

A stereoselective hydroamination transform to access polysubstituted indolizidines.

Stereoselective, intramolecular, formal hydroamination of dienamines via directed hydroboration is reported. Four stereocenters are set in the process. Natural and unnatural indolizidine alkaloids can be synthesized from simple unsaturated amines using the title process.

Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications.

In 1953, Slaughter et al. [D. P. Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] proposed the concept of field cancerization in patients with squamous cell carcinoma of the head and neck (HNSCC) and discussed its clinical significance for the development of second primary tumors and local recurrences. To define the process of field cancerization and its putative clinical implications, we analyzed genetic aberrations in HNSCC and the accompanying macroscopically normal mucosa. In 28 HNSCC patients, loss of heterozygosity was determined in tumor and five noncontiguous mucosal biopsies using eight microsatellite markers at 9p, 3p, and 17p. For patients who showed loss of heterozygosity in their mucosal biopsies, all margins of the surgical specimen were subsequently analyzed to determine the extension of the field. In these cases, additional markers at 8p, 13q, and 18q as well as p53 mutations were included to determine subclonal differences between field and tumor. Genetically altered fields were detected in 36% (10 of 28) of the HNSCC patients. The field varied in size between patients and consisted of genetically different subclones. In 7 of 10 cases, the field extended into the surgical margins. One particular patient with a genetically altered field in a surgical margin developed a local recurrence after 28 months of follow-up. Microsatellite analysis showed that this recurrence had more molecular markers in common with the nonresected premalignant field than with the original tumor, suggesting that this persistent field has progressed further into a new malignancy. Our data show that genetically altered mucosa remains after treatment in a significant proportion of HNSCC patients, which may explain in part the high frequency of local recurrences and second primary tumors. Adequate identification and risk assessment of these genetically altered fields may have profound implications for future patient management.

Molecular assays for the diagnosis of minimal residual head-and-neck cancer: methods, reliability, pitfalls, and solutions.

The prognosis of cancer patients is determined by the radicalness of treatment: residual tumor cells will grow out and develop in manifest local recurrences, regional recurrences, and distant metastases. Classical diagnostic methods such as radiology and histopathology have limited sensitivities, and only by molecular techniques can minimal residual disease be detected. In tissue samples containing the normal tissue counterpart of a tumor, only tumor-specific markers can be exploited, whereas in other samples, tissue-specific markers can be used. At present, there are two main methodologies in use, one based on antigen-antibody interaction and the other based on amplified nucleic acids. The most commonly used nucleic acid markers are mutations or alterations in tumor DNA (tumor-specific markers) or differentially expressed mRNA (tissue-specific markers). Many reports and reviews have been published on the assessment of minimal residual disease by molecular markers, showing either positive or negative clinical correlations. One of the main reasons for these contradictory findings is the technical difficulty in finding the small numbers of tumor cells in the large number of normal cells, which necessitates sensitivities of the assays up to 1 tumor cell in 2 x 10(7) normal cells. These assays often are complex, demand considerable experience, and usually are laborious. In this review, we will address a number of the technical issues related to molecular assays for tumor cell detection that make use of nucleic acids as markers. Many difficulties in data interpretation are at least in part because of technical details that might have been solved by the incorporation of one or more appropriate controls. We hope that this review clarifies a number of these issues and help clinicians and investigators interested in this field to understand and weigh the contradictory findings in the published studies. This will help move the field forward and facilitate clinical implementation.

Comparison of internal dose measures of solvents in breath, blood and urine and genotoxic changes in aircraft maintenance personnel.

Solvents and fuels are in widespread use both in civilian and military populations. 1,1,1-trichloroethane (TCA), xylene, toluene, methyl ethyl ketone (MEK) and methylene chloride are found in a variety of compounds including degreasing agents, paints, coatings, pesticides and paint strippers. Toluene and xylene are also found in fuels, which are complex mixtures of hundreds of agents. The purpose of this investigation was twofold. The first was to determine the optimum medium to measure internal dose of solvents comparing blood, urine and breath. The second was to determine if low level exposures were associated with genotoxic changes after a short-term exposure of fifteen or thirty weeks. To accomplish the first goal a pilot study was initiated involving eight volunteers who worked in aircraft maintenance including sheet metal, painting and assembly mechanic jobs. Industrial hygiene measurements were evaluated over 30 working days. Breath, blood and a 24-hour urine sample were collected twice to compare internal dose parameters. To achieve the second goal, 58 newly hired subjects were monitored prior to exposure and over 30 weeks to determine if there were genotoxic changes as a result of solvent and/or fuel exposure as measured by sister chromatid exchanges (SCEs) and micronuclei (MN). Exposure groups included workers involved in sheet metal (fuel cell) activities, painting, fueling operations and flight line. Results of the pilot study demonstrated that industrial hygiene air samples and internal breath measures taken on the same day were highly correlated for measuring TCA (r = 0.93) and toluene (r = 0.90) but was not as well correlated for the other compounds. Breath measures were more sensitive for measuring low level exposure than were either analytes in blood or 24-hour urine samples; these latter two measures were usually below the limit of detection. A small but statistically significant increase in the frequency of SCEs occurred after 30 weeks of exposure for sheet metal workers (p = 0.003) and for painters (p = 0.05). The MN frequency in the sheet metal workers initially showed a significant increase by 15 weeks, but by 30 weeks had decreased. Chance occurrence of exposures to other occupational or non-occupational agents can not be eliminated as a cause of the genotoxic results since between 58 and 93 total analytes could be found in the breath of some aircraft maintenance personnel.

Polish TC interviews Bill Corey about CCL's work in Africa.

PIP: Natural family planning (NFP) works the same in Cameroon and South Africa as it does elsewhere the author has visited, including Europe, Latin America, and the Pacific Islands. However, one difference observed between cultures is that Africans more easily learn and practice the concept of ecological breast feeding. For the most part, breast feeding has been the only form of birth control practiced in Cameroon, although the government is trying to encourage the use of other contraceptive methods. All of CCL's teaching couples in Cameroon are Black, Catholic, and from the English-speaking section of the country bordering Nigeria. The NFP training program in the city of Kumbo has been very successful and CCL hopes to spread its service everywhere that it can, including to Russia. The author is pleased with CCL's service in Poland.^ieng

Titanium osseointegrated implants combined with hyperbaric oxygen therapy in previously irradiated mandibles.

STATEMENT OF PROBLEM: Treatment for head and neck malignancies commonly involves radiation therapy. As a result of this therapy the vascular supply to irradiated structures is altered and results in decreased tissue perfusion. In addition to vascular changes, bony structures undergo a reduction in osteoblastic and osteoclastic activity. These tissue alterations, especially in the mandible, enhance the risk of osteoradionecrosis. To avoid this occurrence, many patients who have undergone radiation therapy do not receive elective preprosthetic surgeries, including implant therapy. PURPOSE OF STUDY AND METHODS: This report presents the preliminary results of placing 18 titanium screw implants into previously irradiated mandibles in conjunction with hyperbaric oxygen therapy. RESULTS: Of the 18 implants placed, 17 (94%) were judged to be osseointegrated at the abutment connection. One implant did not receive an abutment and was "put to sleep." The remaining 16 (88%) were used for prosthetic rehabilitation. CONCLUSION: The use of implants in irradiated tissues may provide a means of enhancing prosthetic rehabilitation while reducing the risk of tissue trauma that may develop into osteoradionecrosis.

Predictive factors for acute renal failure in thoracic and thoracoabdominal aortic aneurysm surgery.

PURPOSE: The purpose of this study was to analyze the factors associated with acute renal failure in total descending thoracic and thoracoabdominal aortic aneurysm surgery. METHODS: A total of 234 patients underwent thoracoabdominal aortic aneurysm or total descending thoracic aneurysm repair between January 1991 and January 1994. Eighty-five women and 149 men were evaluated. The median age was 67 years (range 8 to 88 years). Seventy-seven patients had type I thoracoabdominal aortic aneurysm, 99 had type II, 51 had type III or IV, and 7 had total descending thoracic aneurysm. Factors such as age, sex, aneurysm type, and visceral and distal aortic perfusion were examined with univariate fourfold table and multivariate logistic regression analysis. RESULTS: Acute renal failure, defined as an increase in serum creatinine by 1 mg/dl per day for two consecutive days after surgery, occurred in 41 (17.5%) of 234 patients. Thirty-six (15%) of 234 patients required dialysis. Twenty (49%) of 41 patients with acute renal failure died. Of the 21 survivors with renal failure, renal failure resolved in 18 (86%) within 30 days of surgery. The univariate odds ratio of death, given acute renal failure, was 6.7 (95% confidence interval [CI] 3.2 to 14.2, p < 0.0001). No significant association was found between the probability of acute renal failure and age, sex, hypertension, right renal artery reattachment, or renal bypass. Factors associated with increased risk of acute renal failure in multivariate analysis were visceral perfusion (odds ratio [OR] = 3.6 95%, CI 1.2 to 11.0, p < 0.02), left renal artery reattachment (OR = 4.4 95%, CI 1.6 to 11.9, p < 0.004), preoperative creatinine > or = 2.8 mg/dl (OR = 10.3, 95% CI 12.0 to 411.8, p < 0.0001), and simple clamp technique (OR = 3.4 95%, CI 1.07 to 10.76, p < 0.04). Direct univariate correlation was seen between preoperative creatinine and acute renal failure (OR = 3.2 per mg/dl increase, 95% CI 2.7 to 10.1, p < 0.0001). CONCLUSION: Postoperative acute renal failure after thoracoabdominal and total descending thoracic aortic aneurysm surgery is associated with preoperative creatinine level, visceral perfusion, left renal artery reattachment, and simple cross-clamp technique.

Foreign body aspiration in dentistry: a review.

This article reviews the dangers of aspirating foreign bodies of dental origin. Two illustrative cases are presented, including an unusual case involving aspiration of an elastomeric impression material. The authors describe the techniques used to identify the foreign body. A radiodensimetric study of four impression materials demonstrates the difficulty of identifying most impression materials. The authors also present some strategies for reducing the risk of aspiration during dental procedures.

Molecular modeling parameters predict antioxidant efficacy of 3-indolyl compounds.

Many dietary constituents, such as indole-3-carbinol, are chemoprotective in toxicity and carcinogenicity bioassays. Indole-3-carbinol and related congeners appear to protect partly via radical and electrophile scavenging. To develop better chemoprotective indoles with lower intrinsic toxicity, we performed molecular graphic and quantum-mechanical analyses of model indolyl compounds to ascertain the determinant molecular features for antioxidant activity. We examined eight structurally related 3-indolyl compounds for relationships between antioxidation potential (using in vitro lipid peroxidation assays) and electronic, polar, and steric parameters, including bond dissociation energies, bond lengths, dipole moments, electronic charge densities, and molecular size parameters. Electronic features of the 3-methylene carbon and 1-nitrogen were not predictive of antioxidative potency due to extensive charge delocalization of the cation radical following electron abstraction from the nitrogen. Antioxidant efficacy of 3-indolyl compounds was most strongly predicted by molecular size parameters and by the energy of electron abstraction as calculated from the difference in heat of formation between the parent compound and its cation radical. A highly predictive multiple linear regression correlation model (r2 = 0.97) was obtained using the parameters of heat of formation, molecular weight, log P, and diplole moment.

Interaction between the Ah receptor and proteins binding to the AP-1-like electrophile response element (EpRE) during murine phase II [Ah] battery gene expression.

We have studied three Phase II genes in the mouse dioxin-inducible [Ah] battery: Nmo1 [encoding NAD(P)H:menadione oxidoreductase], Ahd4 (encoding the cytosolic aldehyde dehydrogenase ALDH3c), and Ugt1*06 (a UDP glucuronosyltransferase). Oxidant-induced Nmo1 gene expression in the c14CoS/c14CoS mouse appears likely to be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on Chr 7 and absence of the enzyme (FAH), which leads to increased levels of endogenous tyrosine oxidative metabolites. We show here that increases in [Ah] Phase II gene expression in the 14CoS/14CoS mouse are correlated with an AP-1-like DNA motif called the electrophile response element (EpRE), which has been found in the 5' flanking regulatory regions of all murine (Ah) Phase II genes. Aromatic hydrocarbon response element (AhREs) are responsible for dioxin-mediated upregulation of all six [Ah] battery genes, and one or more AhREs have been found in the 5' flanking regulatory regions of all of these [Ah] genes. Gel mobility shift assays, with a synthetic oligonucleotide probe corresponding to the EpRE, show that EpRE-binding proteins are more than twice as abundant in 14CoS/14CoS than in the wild-type ch/ch nuclear extracts. Competition studies of EpRE-specific binding with an excess of EpRE, mutated EpRE, AP-1, AhRE3, mutated AhRE3, and C/EBP alpha oligonucleotides suggest that several common transcriptional factors bind to the EpRE and AhRE3 motifs. Two monospecific antibodies to the Ah receptor (AHR) protein block formation of an EpRE-specific complex on gel mobility electrophoresis. These data suggest that AHR (or AHR-related protein) might be an integral part of the EpRE-binding transcriptional complex associated with the oxidative stress response. To our knowledge, this is among the first reports of the same transcription factor operating at two different response elements upstream of a single gene.

Enzyme induction by L-buthionine (S,R)-sulfoximine in cultured mouse hepatoma cells.

Induction of Phase II enzymes of the [Ah] gene battery by L-buthionine (S,R)-sulfoximine (BSO) and other agents was examined in mouse hepatoma Hepa-1c1c7 cells. BSO, a nonelectrophilic inhibitor of gamma-glutamylcysteine synthetase (GCS), is routinely used to examine the toxicological implications of GSH depletion. Exposure to BSO for 24 h produced a 75-85% depletion of GSH levels, proportional to the inhibition of GCS activity, as well as small increases in the UDP-glucuronosyltransferase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activities in Hepa-1 wild-type (wt) cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AHD4) enzyme activities, BSO produced larger increases (110% and 170%, respectively). The mechanisms of NMO1 and AHD4 induction were examined further. In Hepa-1 wt cells, NMO1 and AHD4 activities were increased by the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known inducing agents for these enzymes. However, NMO1 and AHD4 were induced in Ah receptor nuclear translocation-defective mutant (c4) cells by BSO and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produced a concentration-dependent increase in intracellular cysteine levels, but not GSH levels, in the absence or presence of BSO. Furthermore, N-acetylcysteine had no effect on NMO1 activity under any conditions examined, suggesting that GSH levels per se, rather than change in overall thiol status, might be mediating increased NMO1 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[1,2-b]indole in mouse hepatoma cell lines.

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*06, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form A1 in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased gamma-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.