Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an aromatic polyketide synthase.

BACKGROUND: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. RESULTS: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. CONCLUSIONS: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.

Engineering biodiversity with type II polyketide synthase genes.

A very important task in the ongoing search for new clinically useful drugs is the generation of large numbers of structurally diverse compounds. The emerging field of combinatorial biosynthesis, in which nature's chemical capabilities are exploited in a combinatorial 'mix-and-match' fashion, has generated libraries of novel molecules representing great structural diversity which are not available naturally or readily generated through (combinatorial) synthesis. Novel polyketides have been generated by manipulating type II iterative polyketide synthase (PKS) systems that express a variety of combinations of a minimal PKS with ketoreductases, cyclases, and other tailoring enzymes, resulting in a set of design rules to rationally engineer new metabolites. Engineering studies with the Streptomyces coelicolor whiE (spore pigment) and the 'Streptomyces maritimus' enterocin type II PKS provide additional insight on designing diverse assemblies of aromatic, as well as nonaromatic, polyketides.

Significance of the anti-E2 response in self-limited and chronic hepatitis C virus infections in chimpanzees and in humans.

To determine whether there was a correlation between the kinetics or frequency of antibody to mammalian-derived hepatitis C virus (HCV) second envelope protein (E2) and development of chronicity or self-limitation of HCV infections, serial sera were examined for anti-E2, anti-HCV with confirmation with Matrix 2.0 (Abbott Laboratories, Abbott Park, IL), and reverse transcriptase-polymerase chain reaction (RT-PCR) from 6 cases of self-limited infection and 6 cases of chronic infection in chimpanzees, and from 5 cases of self-limited infection and 3 cases of chronic infection in patients. Anti-E2 developed earlier, more frequently, and to higher titer in chimpanzees and patients who were developing chronic infection than in those with self-limited infections. Thus anti-E2 is unlikely to play a role in self-limitation of the infection. However, long-term persistence of anti-E2 correlates with chronic infection. There was little or no correlation between the timing of development of anti-E2 and anti-HCV.

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively.

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra- to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with (18)O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7, 8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes.

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Lack of evidence of hepatitis C infection in 290 blood component recipients, demonstrated by several single-antigen research immunoassays.

BACKGROUND: A group of 290 transfusion recipients enrolled in a prospective study of posttransfusion hepatitis was studied to determine the possibility of previously unrecognized hepatitis C virus (HCV) transmission. STUDY DESIGN AND METHODS: Before and after transfusion, blood specimens that were negative in first-generation enzyme immunoassay (EIA) were tested by current commercial EIAs, several single-antigen research EIAs, and supplemental tests. RESULTS: Current second- and third-generation EIAs identified five subjects (1.7% of total) who had chronic hepatitis C before transfusion. Twenty additional sera had some reactivity with research EIAs. However, those results were the same before and after transfusion (n = 7), had reverted to partially reactive or nonreactive (n = 8), or could not be confirmed by serologic tests or polymerase chain reaction in follow-up specimens (n = 5). CONCLUSIONS: Transient or restricted reactivity to HCV antigens measured by more sensitive research EIAs does not seem to correspond to recent HCV transmission by transfusion. Whether such reactivity could reflect remote HCV infection, with the potential for chronic or intermittent viremia, remains to be determined.

Oleic Acid and Its Positional Isomer, cis-Vaccinic Acid, in the Appendix of Sauromatum guttatum during Anthesis.

The fatty acid profiles of various organs of the thermogenic inflorescence of Sauromatum guttatum and of the sporophylls of thermogenic male cones of two cycad species (Encephalartos ferox and Dioon edule var edule and var angustifolium) were determined by gas chromatography. During anthesis, palmitate (16:0), oleate [18:1 (9)], cis-vaccinate [18:1 (11)], and linoleate [18:2 (9, 12)] were the most abundant fatty acids in the Sauromatum appendix. cis-Vaccinic acid, a positional isomer of oleic acid, was identified by comparing its retention time on a gas chromatography column and its mass spectrum to an authentic compound. The percentage of oleic acid from total fatty acids dropped from about 9 in the morning 3 d before heat production to 6 in the morning 2 d before heat production. At this time, the percentage of cis-vaccinic acid increased from 3 to 11%, and then remained at this level until the inflorescence dried and died. Palmitoleic acid [16:1 (9)], the common precursor of cis-vaccinic acid, is a minor component of total fatty acids. In six other organs of the Sauromatum inflorescence including thermogenic organs, such as male flowers and lower spadix, palmitate, oleate, and linoleate were prevalent but cis-vaccinate was not. The thermogenic male cones of the two cycad species were rich in palmitic, oleic, and linolenic acids. The level of cis-vaccinic acid in these organs was less than 0.5%.

Three new omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus and their biosynthetic interrelationships.

Three minor omega-cycloheptyl fatty acids from Alicyclobacillus cycloheptanicus have been identified as omega-cycloheptylnonanoate, -decanoate, and -alpha-hydroxytridecanoate. The biosynthetic interrelations of these cyclic fatty acids have been studied. 13C-Labeled omega-cycloheptylundecanoate and -alpha-hydroxyundecanoate were converted to omega-cycloheptyldecanoate, which is one carbon shorter in length. alpha-Hydroxylation of omega-cycloheptylundecanate was also observed, but not chain extension to omega-cycloheptyltridecanoate.

Biosynthetic studies on the origin of the cyclohexanecarboxylic acid moiety of ansatrienin A and omega-cyclohexyl fatty acids.

Feeding experiments with [2,6,10,10-2H4]chorismate in Streptomyces collinus (ansatrienin A) and Alicyclobacillus acidocaldarius (omega-cyclohexyl fatty acids), and inhibitor experiments with glyphosate in the latter organism, have shown that the biosynthesis of cyclohexanecarboxylic acid branches off from the shikimate pathway at a point prior to enolpyruvylshikimate 3-phosphate, either at shikimate or shikimate 3-phosphate.

Nursing research: quality of life and perceived health in the elderly.

1. Studies have shown that self-perceived health is significantly related to quality of life for older persons. 2. Terminology regarding quality of life and perceived health was found to be inconsistent in the gerontological nursing research reviewed. 3. Outcomes of the research related to quality of life and perceived health fell into three basic categories: predictors found, relationships found, and no relationships found. 4. More experimental research, more replication of research, more research on older males, more use of nursing theory as a framework for research, and more multidisciplinary gerontological research may contribute useful knowledge to the nursing discipline.

Transcatheter manipulation of asymmetrically opened titanium Greenfield filters.

PURPOSE: The problem of asymmetric opening of the modified hook titanium Greenfield inferior vena cava filter necessitating transcatheter manipulation was evaluated in a retrospective study. PATIENTS AND METHODS: Titanium Greenfield filters were placed in 166 patients over a 36-month period. The radiographic reports of all patients were reviewed to identify cases in which the filter failed to open symmetrically after deployment and catheter or wire manipulation of the filter was performed. The reports and angiograms from these patients were reviewed with respect to the circumstances surrounding filter placement and methods to achieve more symmetric opening. RESULTS: Transcatheter manipulation of asymmetrically opened filters was performed in 15 of 166 cases (9%). In 12 of these patients, acceptable and uneventful opening of the filter was achieved with a guide wire, pigtail catheter, or occlusion balloon catheter. In one case manipulation only partly improved orientation of the limbs, while in another case successful manipulation was complicated by distal migration. In the final case, the asymmetric filter covered only part of the lumen of the vena cava despite manipulations and a second filter was placed for optimal caval interruption. No specific cause for incomplete expansion was identified in any case. CONCLUSION: Marked asymmetry in opening of the modified hook titanium Greenfield filter that warrants manipulation occurs infrequently, but recognition and proper management may be important to ensure optimal caval interruption.

Suspected Marfan Syndrome in a Female Basketball Player.

A group of experts met to discuss a case from the University of California, San Diego, School of Medicine. This case conference is part of a series featuring a variety of sports medicine topics.

The effect of daily Hassles of humoral immunity: an interaction moderated by locus of control.

Research has shown that stress is associated with depressed cellular immune functioning. Controversy exists as to whether humoral immunity shows a similar depression under stress. Several psychological variables have been found to moderate responses to stress. In particular, an internal locus of control has been correlated with better health. The present study examined the level of salivary immunoglobulin A (IgA) and health locus of control in subjects with high and low stress levels. The Hassles scale was administered to identify two subject groups differing significantly in reported stress levels. Results showed no significant differences between the two groups on salivary IgA. There was a significant negative correlation between IgA and internal locus of control. The direction of this correlation is in contrast with previous findings on internality. The evidence suggests that high internal individuals may be especially vulnerable to high levels of stress, particularly stress that they cannot control. Also, there was a significant difference in IgA levels between females depending on whether they were in the first or the second half of their menstrual cycle. Those in the premenstrual phase had lower IgA levels.

Haemophilus influenzae type b disease after rifampin prophylaxis in a day care center: possible reasons for its failure.

Five infants enrolled in a day care center (DCC) developed invasive disease due to Haemophilus influenzae type b during a 25-week period. The isolates from four patients had identical outer membrane proteins and were biotype 1. Although rifampin prophylaxis was recommended on two different occasions, retrospective surveys demonstrated that 39 and 60%, respectively, of infants failed to receive the agent for various reasons. Pharyngeal cultures were obtained from infants, their families and DCC staff after prophylaxis was given the second time. Forty-seven % of DCC infants had positive cultures, and 59% of their households had at least one carrier of the invasive strain. Rifampin prophylaxis administered a third time to infants and members of their households was unsuccessful in eradicating the invasive Haemophilus influenzae type b strain from DCC infants. The possible reasons for failure of rifampin in this DCC outbreak included incomplete understanding by physicians and health department officials of the factors affecting a DCC outbreak of Haemophilus influenzae type b disease and failure properly to implement rifampin prophylaxis to all contacts and the index cases.

Breed-specific dog hypersensitivity in humans.

The contribution of allergenic components of dander and serum from individual dog breeds in human hypersensitivity and the different responses to the various breeds in vivo and in vitro were examined. Differences in IgE antibodies directed against 13 individual dog breed danders and five different breed-specific sera were measured in groups sensitive and nonsensitive to dogs by the radioallergosorbent test (RAST). Cross-reactivity between dander extracts and sera was examined by RAST inhibition. Mean RAST binding to dog danders and sera was 15.9% and 14%, respectively, for the dog-sensitive group versus 0.6% for both danders and sera in the nonsensitive group. The mean difference in binding between groups was statistically different for all dander extracts and sera. The variances in binding were statistically greater for danders than sera in six of eight dog-reactive subjects. RAST binding and intradermal skin tests at a 1:10,000 w/v dilution correlated well (p < 0.05) for seven of nine breed-specific extracts. RAST inhibition studies showed that dog serum proteins were relatively poor inhibitors of dander extracts, and vice versa. Variability of skin test and RAST responses to different dog breed dander extracts support the existence of breed-specific allergens.