Presumed primary cardiac mast cell tumor, pericardial effusion, and arrhythmia in a dog.

A 13-year-old female spayed border collie cross presented for pericardial effusion, arrhythmia, and a suspected cardiac mass. Echocardiogram revealed severe thickening and hypokinesis of the interventricular septum with a heterogenous, cavitated myocardium, concerning for neoplasia. Electrocardiogram revealed predominantly accelerated idioventricular rhythm with frequent periods of nonsustained ventricular tachycardia. Occasional prolonged PR intervals terminating in an aberrantly conducted QRS complex were present. These beats were postulated to represent either first-degree atrioventricular block with aberrant QRS conduction or atrioventricular dissociation. Cytology of the pericardial effusion revealed atypical, suspected neoplastic, mast cells. The patient was euthanized, and postmortem examination confirmed full-thickness infiltration of the interventricular septum by a mast cell tumor, with metastasis to the tracheobronchial lymph node and spleen. Given the anatomic location of the mass, the observed atrioventricular nodal conduction delay may represent neoplastic infiltration of the atrioventricular node. Neoplastic infiltration of the ventricle was suspected to cause the accelerated idioventricular rhythm and ventricular tachycardia. To the authors' knowledge, this is the first reported case of a primary cardiac mast cell tumor causing arrhythmia and pericardial effusion in a dog.

Developmentally regulated rpd3p homolog specific to the transcriptionally active macronucleus of vegetative Tetrahymena thermophila.

A clear relationship exists between histone acetylation and transcriptional output, the balance of which is conferred by opposing histone acetyltransferases (HATs) and histone deacetylases (HDACs). To explore the role of HDAC activity in determining the transcriptional competency of chromatin, we have exploited the biological features of Tetrahymena as a model. Each vegetative cell contains two nuclei: a somatic, transcriptionally active macronucleus containing hyperacetylated chromatin and a transcriptionally silent, germ line micronucleus containing hypoacetylated histones. Using a PCR-based strategy, a deacetylase gene (named THD1) encoding a homolog of the yeast HDAC Rpd3p was cloned. Thd1p deacetylates all four core histones in vitro. It resides exclusively in the macronucleus during vegetative growth and is asymmetrically distributed to developing new macronuclei early in their differentiation during the sexual pathway. Together, these data are most consistent with a potential role for Thd1p in transcriptional regulation and suggest that histone deacetylation may be important for the differentiation of micronuclei into macronuclei during development.

A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila.

Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins is Drosophila HP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624-13629, 1998). Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (DeltaHHP1). However, during a shift to nongrowth conditions, the survival rate of DeltaHHP1 cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in DeltaHHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response.

An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena.

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Extra telomeres, but not internal tracts of telomeric DNA, reduce transcriptional repression at Saccharomyces telomeres.

Yeast telomeric DNA is assembled into a nonnucleosomal chromatin structure known as the telosome, which is thought to influence the transcriptional repression of genes placed in its vicinity, a phenomenon called telomere position effect (TPE). The product of the RAP1 gene, Rap1p, is a component of the telosome. We show that the fraction of cells exhibiting TPE can be substantially reduced by expressing large amounts of a deletion derivative of Rap1p that is unable to bind DNA, called Rap1 delta BBp, or by introducing extra telomeres on a linear plasmid, presumably because both compete in trans with telomeric chromatin for factor(s) important for TPE. This reduction in TPE, observed in three different strains, was demonstrated for two different genes, each assayed at a different telomere. In contrast, the addition of internal tracts of telomeric DNA on a circular plasmid had very little effect on TPE. The product of the SIR3 gene, Sir3p, appears to be limiting for TPE. Overexpression of Sir3p completely suppressed the reduction in TPE observed with expression of Rap1 delta BBp, but did not restore high levels of TPE to cells with extra telomeres. These results suggest that extra telomeres must titrate a factor other than Sir3p that is important for TPE. These results also provide evidence for a terminus-specific binding factor that is a factor with a higher affinity for DNA termini than for nonterminal tracts of telomeric DNA and indicate that this factor is important for TPE.

Cefuroxime treatment failure of nontypable Haemophilus influenzae meningitis associated with alteration of penicillin-binding proteins.

A 10-year-old boy presented with nuchal rigidity and cerebrospinal fluid (CSF) leukocytosis initially and again on day 6 of intravenous cefuroxime therapy (200 mg/kg/day). Both CSF specimens yielded nontypable beta-lactamase-negative Haemophilus influenzae that were susceptible by disk tests but relatively resistant to cefuroxime (MIC, 8- to 16-fold greater than that of control isolates). To define the mechanism of resistance, the cefuroxime resistance marker was transformed to a susceptible H. influenzae recipient; inactivation and permeability of beta-lactam substrate were tested and the penicillin-binding protein (PBP) profiles were examined. Inactivation of beta-lactam substrate was not detected and reduced permeability was not found. However, reduced beta-lactam binding to PBPs 4 and 5 was observed; 18- to 27-fold more penicillin and 2.5-to 4-fold more cefuroxime was required to saturate or block 50% of the binding sites of these PBPs, respectively. Thus, reduced affinity of PBPs 4 and 5 for beta-lactam substrate appears to be the mechanism of cefuroxime resistance in this strain. The reduced affinity of these targets appears to have contributed to the bacteriologic and clinical failure in this patient.

Problems with current recommendations for susceptibility testing of Haemophilus influenzae.

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.