Citations

Abstract Species in the genus Pneumocystis can cause severe pneumonia in immune-compromised hosts. The identification of specific targets present in Pneumocystis species, but lacking in mammalian hosts, is paramount to developing new means to treat this infection. One such potential protein is Rtt109, which is a type of histone acetyltransferase (HAT) required for DNA replication in fungi, but not found in mammals. Sequence orthologues of Rtt109 are present in other fungi, but are absent in mammals, making it a potential pan-specific target against medically relevant fungi. Accordingly, we sought to identify the presence of an Rtt109 in P. carinii. A Pneumocystis carinii (Pc) Rtt109 165-bp partial sequence was initially identified from the incomplete P. carinii genome database. Subsequently, a full-length, 1,128-bp cDNA with homology to Saccharomyces cerevisiae Rtt109 (39% Basic Local Alignment Search Tool (BLASTP)) was cloned and characterized. Sequence analysis of PcRtt109 indicated that the P. carinii molecule contains the putative catalytic aspartate present in yeast. We further demonstrated that the PcRtt109 expressed in rtt109Δ S. cerevisiae cells restored H3-K56 acetylation and the sensitivity toward DNA-damaging agents of rtt109Δ mutant cells. Purified PcRtt109 had the ability to acetylate lysine-56 of histone H3, similar to the ability of Schizosaccharomyces pombe Rtt109 protein. The site-directed mutagenesis of PcRtt109 D84A, a potential regulatory site in the Rtt109 HAT family, abolished H3 acetylation, whereas a DD218/219AA mutation that compromised the activity of ScRtt109 had little effect, demonstrating similarities and differences in Pneumocystis PcRtt109 compared with yeast Saccharomyces cerevisiae Rtt109. These results indicate that P. carinii contains an Rtt109 HAT molecule, and represent the complete identification and characterization of a HAT molecule from this important opportunistic fungal pathogen.

Object A challenge associated with deep brain stimulation (DBS) in treating advanced Parkinson disease (PD) is the direct visualization of brain nuclei, which often involves indirect approximations of stereotactic targets. In the present study, the authors compared T2*-weighted images obtained using 7-T MR imaging with those obtained using 1.5- and 3-T MR imaging to ascertain whether 7-T imaging enables better visualization of targets for DBS in PD. Methods The authors compared 1.5-, 3-, and 7-T MR images obtained in 11 healthy volunteers and 1 patient with PD. Results With 7-T imaging, distinct images of the brain were obtained, including the subthalamic nucleus (STN) and internal globus pallidus (GPi). Compared with the 1.5- and 3-T MR images of the STN and GPi, the 7-T MR images showed marked improvements in spatial resolution, tissue contrast, and signal-to-noise ratio. Conclusions Data in this study reveal the superiority of 7-T MR imaging for visualizing structures targeted for DBS in the management of PD. This finding suggests that by enabling the direct visualization of neural structures of interest, 7-T MR imaging could be a valuable aid in neurosurgical procedures.

Abstract Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17β-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P < 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P < 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) and other idiopathic interstitial pneumonias (IIPs) have similar clinical and radiographic features, but their histopathology, response to therapy, and natural history differ. A surgical lung biopsy is often required to distinguish between these entities. Objectives We sought to determine if clinical variables could predict a histopathologic diagnosis of IPF in patients without honeycomb change on high-resolution computed tomography (HRCT). Methods Data from 97 patients with biopsy-proven IPF and 38 patients with other IIPs were examined. Logistic regression models were built to identify the clinical variables that predict histopathologic diagnosis of IPF. Measurements and Main Results Increasing age and average total HRCT interstitial score on HRCT scan of the chest may predict a biopsy confirmation of IPF. Sex, pulmonary function, presence of desaturation, or distance walked during a 6-minute walk test did not help discriminate pulmonary fibrosis from other IIPs. Conclusions Clinical data may be used to predict a diagnosis of IPF over other IIPs. Validation of these data with a prospective study is needed.

Rewarming patients after profound hypothermia may result in acute heart failure and high mortality (50–80%). However, the underlying pathophysiological mechanisms are largely unknown. We characterized cardiac contractile function in the temperature range of 15–30°C by measuring the intracellular Ca2+concentration ([Ca2+]i) and twitch force in intact left ventricular rat papillary muscles. Muscle preparations were loaded with fura-2 AM and electrically stimulated during cooling at 15°C for 1.5 h before being rewarmed to the baseline temperature of 30°C. After hypothermia/rewarming, peak twitch force decreased by 30–40%, but [Ca2+]iwas not significantly altered. In addition, we assessed the maximal Ca2+-activated force (Fmax) and Ca2+sensitivity of force in skinned papillary muscle fibers. Fmaxwas decreased by ∼30%, whereas the pCa required for 50% of Fmaxwas reduced by ∼0.14. In rewarmed papillary muscle, both total cardiac troponin I (cTnI) phosphorylation and PKA-mediated cTnI phosphorylation at Ser23/24 were significantly increased compared with controls. We conclude that after hypothermia/rewarming, myocardial contractility is significantly reduced, as evidenced by reduced twitch force and Fmax. The reduced myocardial contractility is attributed to decreased Ca2+sensitivity of force rather than [Ca2+]iitself, resulting from increased cTnI phosphorylation.

Abstract Rationale Debate exists about the immunogenicity and protective efficacy of antibodies produced by the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in chronic obstructive pulmonary disease (COPD). The 7-valent diphtheria-conjugated pneumococcal polysaccharide vaccine (PCV7) induces a more robust immune response than PPSV23 in healthy elderly adults. Objectives We hypothesized that serotype-specific IgG antibody concentration and functional antibody activity would be superior after PCV7 vaccination compared with PPSV23 in moderate to severe COPD. We also posited that older age and prior PPSV23 vaccination would be associated with reduced vaccine responsiveness. Methods One hundred twenty patients with COPD were randomized to PPSV23 (63 subjects) or PCV7 (57 subjects). IgG concentrations were determined by ELISA; functional antibody activity was assayed with a standardized opsonophagocytosis assay and reported as an opsonization killing index (OPK). Increases in serotype-specific IgG and OPK at 1 month post vaccination were compared within and between vaccine groups. Measurements and Main Results Both vaccines were well tolerated. Within each study group, postvaccination IgG and OPK were higher than baseline (P < 0.01) for all serotypes. Adjusted for baseline levels, postvaccination IgG was higher in the PCV7 group than the PPSV23 group for all seven serotypes, reaching statistical significance for five (P < 0.05). PCV7 resulted in a higher OPK for six of seven serotypes (statistically greater for four) compared with PPSV23. In multivariate analyses, younger age, vaccine naivety, and receipt of PCV7 were associated with increased OPK responses. Conclusions PCV7 induces a superior immune response at 1 month post vaccination compared with PPSV23 in COPD. Older age and prior PPSV23 reduce vaccine responsiveness. Clinical trial registered with www.clinicaltrials.gov (NCT00457977).

Mutation in leucine-rich repeat kinase-2 ( LRRK2 ) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.