The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont "Candidatus Endobugula sertula." In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-"E. sertula" association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.

This longitudinal study investigated the relationship between denial of illness and the course of recovery in patients with coronary heart disease. Using a newly developed interview instrument, the Levine Denial of Illness Scale (LDIS), the level and modes of denial were assessed in 45 male patients who were hospitalized for myocardial infarction or for coronary bypass surgery, of whom 30 were followed for 1 year after discharge. The reliability, internal consistency, and validity of the LDIS were found to be satisfactory. Furthermore, the LDIS showed discriminant validity from trait measures of denial. LDIS scores were not associated with severity of illness or risk factors. High deniers spent fewer days in intensive care and had fewer signs of cardiac dysfunction during their hospitalization relative to low deniers. However, in the year following discharge, high deniers adapted more poorly than low deniers: high deniers were more noncompliant with medical recommendations and required more days of rehospitalization. The findings suggest that denial of illness is adaptive during acute hospital recovery, but is maladaptive in the long-run after hospital discharge.

Although CYP2C8, CYP2C9, and CYP2C19 play an important role in drug biotransformation, factors influencing the expression and activity of these CYP2C P450s in human liver remain largely undefined. We used primary cultures of human hepatocytes from 15 subjects to assess the inducibility of CYP2C enzyme expression by prototypical inducer agents, including rifampicin, dexamethasone, and phenobarbital. After culture for 72 h in serum-free medium on collagen, Western blotting revealed that CYP2C9 was the only CYP2C enzyme expressed at appreciable levels in untreated hepatocytes. Subsequent treatment with 25 M rifampicin for 48 h elicited marked increases in CYP2C8 (700 Ϯ 761%), CYP2C19 (854%), and CYP2C9 (209 Ϯ 176%) protein content versus a 550 Ϯ 170% enhancement of CYP3A4 enzyme levels. Parallel increases in CYP2C mRNAs, measured by Northern blotting and/or RNase protection, were found in rifampicintreated hepatocytes, with CYP2C8, CYP2C9, and CYP2C19 transcripts exhibiting increases of 688 Ϯ 635, 207 Ϯ 49, and 230 Ϯ 60%, respectively, versus an 8.8-fold enhancement of CYP3A4 mRNA levels. Dexamethasone (10 M) treatment enhanced CYP2C8 mRNA (360 Ϯ 100%) and protein (274%) content, although this steroid had less effect on CYP2C9 and CYP2C19 transcripts (23 Ϯ 21% and 21 Ϯ 36%, respectively) and enzyme levels (55 and 143%, respectively). Phenobarbital (100 M) was a powerful inducer of CYP2C9 (850%) and CYP2C19 (735%) mRNA content, and also increased CYP2C8 (610%) and CYP3A4 (205%) transcripts. Our results show that CYP2C enzyme expression in human hepatocytes is highly inducible by rifampicin, dexamethasone, and phenobarbital. Because these xenobiotics are ligands and/or activators of the pregnane X receptor and/or constitutive androstane receptor, such orphan nuclear receptors and their response elements may partake in regulating CYP2C gene expression in humans.

"Candidatus Endobugula sertula," the uncultivated bacterial symbiont of Bugula neritina, is the proposed source of the bryostatin family of anticancer compounds. We cloned a large modular polyketide synthase (PKS) gene complex from "Candidatus Endobugula sertula" and characterized one gene, bryA, which we propose is responsible for the initial steps of bryostatin biosynthesis. Typical PKS domains are present. However, acyltransferase domains are lacking in bryA, and beta-ketoacyl synthase domains of bryA cluster with those of PKSs with discrete, rather than integral, acyltransferases. We propose a model for biosynthesis of the bryostatin D-lactate starter unit by the bryA loading module, utilizing atypical domains homologous to FkbH, KR, and DH. The bryA gene product is proposed to synthesize a portion of the pharmacologically active part of bryostatin and may be useful in semisynthesis of clinically useful bryostatin analogs.