Technique to manage intraoperative cuff leak from damaged endotracheal tube pilot balloon.

We report a technique that was utilized to manage an intraoperative airway complication occurring during orthognathic surgery wherein the endotracheal tube pilot balloon was inadvertently damaged during the procedure. Readily available operating room materials were used to safely and rapidly repair the damaged endotracheal tube pilot balloon. This allowed the perioperative team to avoid emergent endotracheal tube exchange and potential airway complications.

Green tea polyphenols for prostate cancer chemoprevention: a translational perspective.

Every year nearly 200,000 men in the United States are diagnosed with prostate cancer (PCa), and another 29,000 men succumb to the disease. Within certain regions of the world population based studies have identified a possible role for green tea in the prevention of certain cancers, especially PCa. One constituent in particular, epigallocatechin-3-gallate also known as EGCG has been shown in cell culture models to decrease cell viability and promote apoptosis in multiple cancer cell lines including PCa with no effect on non-cancerous cell lines. In addition, animal models have consistently shown that standardized green tea polyphenols when administered in drinking water delay the development and progression of PCa. Altogether, three clinical trials have been performed in PCa patients and suggest that green tea may have a distinct role as a chemopreventive agent. This review will present the available data for standardized green tea polyphenols in regard to PCa chemoprevention that will include epidemiological, mechanism based studies, safety, pharmacokinetics, and applicable clinical trials. The data that has been collected so far suggests that green tea may be a promising agent for PCa chemoprevention and further clinical trials of participants at risk of PCa or early stage PCa are warranted.

Recombinant bacteria for mosquito control.

Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.

Verdoheme reactivity. Remarkable paramagnetically shifted (1)H NMR spectra of intermediates from the addition of hydroxide or methoxide with Fe(II) and Fe(III) verdohemes.

Studies of the reaction of 5-oxaporphyrin iron complexes (verdohemes) with methoxide ion or hydroxide ion have been undertaken to understand the initial step of ring opening of verdohemes. High-spin [ClFe(III)(OEOP)] undergoes a complex series of reactions upon treatment with hydroxide ion in chloroform, and similar species are also detected in dichloromethane, acetonitrile, and dimethyl sulfoxide. Three distinct paramagnetic intermediates have been identified by (1)H NMR spectroscopy. These reactive species are formed by addition of hydroxide to the macrocycle and to the iron as an axial ligand. Treatment of low-spin [(py)(2)Fe(II)(OEOP)]Cl (OEOP is the monoanion of octaethyl-5-oxaporphyrin) with excess methoxide ion in pyridine solution produces [(py)(n)()Fe(II)(OEBOMe)] (n = 1 or 2) ((OEBOMe), dianion of octaethylmethoxybiliverdin), whose (1)H NMR spectrum undergoes marked alteration upon addition of further amounts of methoxide ion. An identical (1)H NMR spectrum, which is characterized by methylene resonances with both upfield and downfield paramagnetic shifts, is formed upon treatment of [Fe(II)(OEBOMe)](2) with methoxide in pyridine solution and results from the formation of [(MeO)Fe(II)(OEBOMe)](-).

p21 gene regulation during enterocyte differentiation.

BACKGROUND: Enterocyte differentiation is associated with a withdrawal from the cell cycle and the transcriptional activation of the cell cycle inhibitor, p21. We sought to define the molecular mechanisms involved in p21 gene activation in an in vitro system. METHODS: Transient transfections were performed in HT-29 cells with plasmids containing various 5' deletions of the p21 promoter upstream of the luciferase reporter -/+ the histone deacetylase 1 (HDAC1) expression plasmid. After 24 h, cells were treated -/+ 5 mM sodium butyrate (NaBu) or another histone hyperacetylating agent, trichostatin A (TSA, 0.3 microM) for 24 h. After protein extraction, luciferase activity was measured. Acid/urea/triton gel electrophoresis was performed to examine histone acetylation in cells. RESULTS: NaBu and TSA both caused histone H4 hyperacetylation. Both NaBu and TSA caused a marked increase in the transactivation of plasmids containing 291 bp of the p21 promoter upstream of the transcriptional start site, similar to that previously seen for a 2.4-kb construct. A decrease in reporter gene induction was seen between 173 and 153 bp. This was followed by a marked increase in promoter induction from 143 to 117 bp. Finally, only low basal activity was seen in the case of the 93-bp plasmid. HDAC1 blocked NaBu-mediated induction of all plasmids. CONCLUSIONS: p21 gene activation during HT-29 cell differentiation occurs via at least two regions of cis-acting elements: one located between -93 and -117 bp, and the other between -173 and -291 bp. Histone hyperacetylation likely plays a role in this activation.

Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence.

Previously we demonstrated that the yield of Cry3A (70 kDa) can be increased as much as 10-fold when cry3A including its upstream STAB-SD mRNA stabilizing sequence is expressed in Bacillus thuringiensis under the control of cyt1A promoters. To determine whether the cyt1A promoters/STAB-SD combination (cyt1AP/STAB) has broader applicability, we used it to synthesize two other Cry endotoxins in the 70-kDa mass range, Cry2A and Cry11A. Combination of cyt1AP/STAB with orfs 2 and 3 of the cry2A operon yielded about 4. 4-fold the amount of Cry2A obtained with the wild-type cry2A operon. The yield of Cry11A obtained with a construct that contained the cyt1AP/STAB, cry11A and the 20-kDa protein gene was 1.3-fold the amount obtained with a construct similar to the wild-type operon. These results demonstrate that the cyt1AP/STAB combination can enhance synthesis of different Cry proteins significantly, but that the level of enhancement varies with the specific protein synthesized.

HLA-class II genes modify outcome of Toxoplasma gondii infection.

Associations between Human Leukocyte Antigen (HLA) (i.e. human major histocompatibility complex [MHC]) genes and susceptibility to infections and inflammatory processes have been described, but causal relationships have not been proven. We characterized effects of HLA-DQ alleles on outcome of congenital toxoplasma infection and found that among Caucasians, the DQ3 gene frequency was significantly higher in infected infants with hydrocephalus (0.783) than infected infants without hydrocephalus (0.444) or published normal controls (0.487). We then developed a novel animal model to definitively determine the effect of these HLA DQ molecules on the severity of toxoplasmosis. Human MHC-Class II transgenes reduced parasite burden and necrosis in brains of mice infected with Toxoplasma gondii. Consistent with the observed association between DQ3 and hydrocephalus in human infants, in the murine model the DQ3(DQ8; DQB1*0302) gene protected less than DQ1 (DQ6; DQB1*0601). Our findings definitively prove a cause and effect relationship between human MHC genes and resistance to infection, provide novel means to characterise human immune responses that are protective or pathogenic in infections, and are important for vaccine development.

Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase.

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 microM in 2 days and effectively eliminates the parasite in 2-4 days at 10-100 microM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.

Evidence for the shikimate pathway in apicomplexan parasites.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

Effects of substituting granulin or a granulin-polyhedrin chimera for polyhedrin on virion occlusion and polyhedral morphology in Autographa californica multinucleocapsid nuclear polyhedrosis virus.

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 micron) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.

Grief support for nursing staff in the ICU.

Patient death is a stressful experience for the patient, family, and the healthcare team. Nurses-often have only informal resources for coping with the sadness and grief they might experience. Realizing the need for nursing grief support, a group of staff nurses from the intensive care unit formed a grief support group. Using information from the literature and critical incident stress debriefing, the group developed support interventions to aid intensive care unit staff after patient death.

Type IV collagen is detectable in most, but not all, basement membranes of Caenorhabditis elegans and assembles on tissues that do not express it.

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode alpha1- and alpha2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface-associated molecule(s) is required to facilitate type IV collagen assembly.

Bronchial carcinogenesis in syngeneic hamsters.

Endobronchial sustained release implants of carcinogen were placed in males (m) and females (f) of four varieties of syngeneic hamsters: BIOF1D; BIO87.20; BIO1.5; BIO15.16. The sequential progression of carcinogenesis that occurred was faster for 1.5m than for 1.5f (P = 0.01) and less rapid for 15.16m than for 87.20m and F1Dm (P < 0.05). Fewer invasive cancers occurred in 15.16m than in the other male varieties (P < 0.01), in 1.5m than in 87.20m (P < 0.05), and in 87.20f than in 87.20m (P < 0.05). Adenocarcinoma occurred with greater frequency in the 1.5 variety than in the F1D variety (P < 0.05). Significant variability in susceptibility, incidence, and types of invasive cancers formed exists, providing new opportunities for further study of bronchial carcinogenesis.

Perceived determinants of risk for breast cancer and the relations among objective risk, perceived risk, and screening behavior over time.

Interrelations among perceived risk for breast cancer, objective risk factors, and both breast self-examination (BSE) and mammography screening were examined across two waves of a longitudinal study of breast cancer screening. Participants were a community sample of 335 predominantly White middle-class women, aged 37 to 77, who had not had breast cancer. Factors believed by women to determine their self-rated risk level for breast cancer were investigated. Women held optimistic biases about their own breast cancer risk; they often erroneously attributed their relatively lower perceived risk to personal actions, including mammography screening. Compliance with mammography screening but not BSE recommendations increased over time. Perceived susceptibility to breast cancer was related to both family history and breast symptomatology; early mammography screening was positively related to perceived susceptibility later in time.

Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis.

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1. In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni is due to synergism among three or four of their major proteins.

Analysis of mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans.

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxy-terminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.

Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene.

The nematode Caenorhabditis elegans has two type IV collagen genes homologous to the mammalian alpha 1(IV) and alpha 2(IV) collagen genes. We demonstrate by transgenic rescue of mutant animals that the genetic locus encoding the C. elegans alpha 2(IV) collagen gene is let-2 on the X chromosome. The most severe effect of mutations in let-2 is temperature-sensitive embryonic lethality. The embryonic lethal phenotype is similar to that seen in animals with mutations in the alpha 1(IV) collagen gene, emb-9. The sequence of the entire C. elegans alpha 2(IV) collagen gene is presented. Comparisons with mammalian type IV collagen sequences show high amino acid sequence conservation in the C-terminal NCl domain and of crosslinking residues (Cys and Lys) in the N-terminal 7S domain. RT-PCR analysis shows that transcripts of the C. elegans alpha 2(IV) collagen gene are alternatively spliced. Transcripts contain one of two mutually exclusive exons, exon 9 or 10. These exons encode very similar products, differing primarily in the sequence of a 9-10 amino acid Gly-X-Y interruption. The expression of these alternatively spliced alpha 2(IV) collagen transcripts is developmentally regulated. In embryos over 90% of the alpha 2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. This shift in expression of alternative alpha 2(IV) collagen transcripts suggests that C. elegans embryos may require a different form of alpha 2(IV) collagen than do larvae and adults.

The 3' regulatory region of the Abdominal-B gene: genetic analysis supports a model of reiterated and interchangeable regulatory elements.

The Abdominal-B (Abd-B) gene is one of three genes in the bithorax complex, a cluster of homeotic genes in Drosophila. During embryogenesis Abd-B is expressed in a complex pattern, producing four different transcript classes, each of which exhibits a unique spatial pattern of expression. Proper regulation of the class A transcripts is required for appropriate development of the fifth through eighth abdominal segments and is mediated, in part, by a 60-kb regulatory region located 3' of the gene. We have isolated a new mutation, designated Abd-BCorset, which is caused by a deletion that leaves 15 kb of the 3' regulatory sequences immediately adjacent to the gene, but removes 45 kb of the more distant 3' regulatory elements. This mutation produces an unexpected homeotic segmental transformation of the fourth through seventh abdominal segments, and has been analyzed by genetic and molecular techniques. In situ hybridization to Abd-BCorset embryos shows a uniform and moderate level of the Abd-B class A transcript in the posterior abdomen, rather than the normal graded pattern of expression. Our analysis of the Abd-BCorset mutation has prompted a model of the 3' regulatory region of Abd-B based on reiterated cell type-specific elements controlled by adjacent position-sensitive activating elements. The gradient of Abd-B expression normally observed in the posterior abdomen appears to be achieved by varying the number of reiterated elements that are active in each segment.