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Samuel Kaplan

Publications and source records attributed to Samuel Kaplan.

At least 19 recordsLinked to original sources
PubMed2006

Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes.

The analysis of proteomes from aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 cell cultures by liquid chromatography-mass spectrometry yielded approximately 6,500 high confidence peptides representing 1,675 gene products (39% of the predicted proteins). The identified proteins corresponded primarily to open reading frames (ORFs) contained within the two chromosomal elements of this bacterium, but a significant number were also observed from ORFs associated with 5 naturally occurring plasmids. Using the accurate mass and time (AMT) tag approach, comparative studies showed that a number of proteins were uniquely detected within the photosynthetic cell culture. The estimated abundances of proteins observed in both aerobic respiratory and photosynthetic grown cultures were compared to provide insights into bioenergetic models for both modes of growth. Additional emphasis was placed on gene products annotated as hypothetical to gain information as to their potential roles within these two growth conditions. Where possible, transcriptome and proteome data for R. sphaeroides obtained under the same culture conditions were also compared.

Aerobiosis↗
PubMed2006

Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures.

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.

Aerobiosis↗
PubMed2006

Interacting specificity of a histidine kinase and its cognate response regulator: the PrrBA system of Rhodobacter sphaeroides.

Using a yeast two-hybrid assay system, it was demonstrated that the four-helix bundle of the Rhodobacter sphaeroides PrrB histidine kinase both serves as the interaction site for the regulatory domain of its cognate response regulator PrrA and is the primary determinant of the interaction specificity. The alpha-helix 1 and its flanking turn region within the dimerization domain (DD) of the PrrB histidine kinase appear to play an important role in conferring the recognition specificity for the PrrA response regulator on the DD. The catalytic ATP-binding domain of the histidine kinase, which functions as the catalytic unit for the phosphotransfer reaction from ATP to the conserved histidine residue in the DD, also appears to contribute to the enhancement of the recognition specificity conferred by the DD. It was also revealed that replacement of Asp-63 and Lys-113 of the PrrA response regulator by alanine abolished protein-protein interactions between PrrA and its cognate histidine kinase PrrB, whereas mutations of Asp-19, Asp-20 and Thr-87 to alanine did not affect protein-protein interactions, indicating that among the active site residues of PrrA, Asp-63 and Lys-113 are important not only in the function of PrrA but also for protein-protein interactions between PrrA and PrrB.

Amino Acid Sequence↗
PubMed2005

Mild dilated cardiomyopathy and increased left ventricular mass predict mortality: the prospective P2C2 HIV Multicenter Study.

BACKGROUND: Many HIV-infected children die with cardiac abnormalities. We sought to understand the course of these HIV-associated abnormalities and their impact on all-cause mortality. METHODS: We describe longitudinal changes in left ventricular (LV) structure and function and mortality in 185 children vertically infected with HIV. Serial cardiac data were obtained from 0.1 to 10 years of age. Age- or body surface area-adjusted z scores were calculated for 10 echocardiographic outcomes. RESULTS: Median age at first echocardiogram was 2 years (range 0.2-9.4 years); median follow-up was 3.6 years (range 0-6.3 years). The 5-year cumulative incidence of congestive heart failure was 12.3%. Mean fractional shortening z scores declined from -0.65 at 1 year of age to -1.47 at 3 years of age without further decline between 3 and 10 years of age. Among children with 2 echocardiograms performed in the first year of follow-up, mild LV dysfunction (fractional shortening of < -2 SD on both echocardiograms) was present in 29 (18%) of 158 children. For these 29 children, the 5-year mortality was 55.4%. Left ventricular mass z scores were elevated at 1 year (mean z score 0.68, P < .001) and remained elevated throughout follow-up. In the 8 children with LV mass z score of > 2 SD on both initial and follow-up echocardiograms, the 5-year mortality was 75%. CONCLUSION: In HIV-infected children, LV structure and function progressively deteriorated in the first 3 years of life, resulting in subsequent persistent mild LV dysfunction and increased LV mass. Chronic mild depression of LV function and elevated LV mass were associated with higher all-cause mortality.

Cardiomyopathy, Dilated↗
PubMed2005

Combining microarray and genomic data to predict DNA binding motifs.

The ability to detect regulatory elements within genome sequences is important in understanding how gene expression is controlled in biological systems. In this work, microarray data analysis is combined with genome sequence analysis to predict DNA sequences in the photosynthetic bacterium Rhodobacter sphaeroides that bind the regulators PrrA, PpsR and FnrL. These predictions were made by using hierarchical clustering to detect genes that share similar expression patterns. The DNA sequences upstream of these genes were then searched for possible transcription factor recognition motifs that may be involved in their co-regulation. The approach used promises to be widely applicable for the prediction of cis-acting DNA binding elements. Using this method the authors were independently able to detect and extend the previously described consensus sequences that have been suggested to bind FnrL and PpsR. In addition, sequences that may be recognized by the global regulator PrrA were predicted. The results support the earlier suggestions that the DNA binding sequence of PrrA may have a variable-sized gap between its conserved block elements. Using the predicted DNA binding sequences, a whole-genome-scale analysis was performed to determine the relative importance of the interplay between the three regulators PpsR, FnrL and PrrA. Results of this analysis showed that, compared to the regulation by PpsR and FnrL, a much larger number of genes are candidates to be regulated by PrrA. The study demonstrates by example that integration of multiple data types can be a powerful approach for inferring transcriptional regulatory patterns in microbial systems, and it allowed the detection of photosynthesis-related regulatory patterns in R. sphaeroides.

Bacterial Proteins↗
PubMed2004

Reconstitution of the Rhodobacter sphaeroides cbb3-PrrBA signal transduction pathway in vitro.

The PrrBA two-component system in Rhodobacter sphaeroides 2.4.1, which is composed of the PrrB histidine kinase and the PrrA response regulator, controls the expression of all of the photosynthesis genes, either directly or indirectly, in response to changes in oxygen tension. In vivo under aerobic conditions it is the cbb(3) cytochrome c oxidase which generates an inhibitory signal preventing the accumulation of activated PrrA. Using purified cbb(3) cytochrome c oxidase, PrrB, and PrrA, we demonstrate in vitro that the cbb(3) oxidase inhibits PrrB activity by apparently increasing the intrinsic PrrB phosphatase activity, which dephosphorylates phosphorylated PrrA without alteration of the PrrB kinase activity. The transmembrane domain of PrrB is required for the enhancement of PrrB phosphatase activity by the cbb(3) oxidase. Full-length PrrB has a significantly greater ability to phosphorylate PrrA than does truncated PrrB lacking the transmembrane domain. This is at least in part due to the lower autophosphorylation rate of the truncated PrrB relative to the full-length PrrB. This finding provides evidence that the sensing domain (transmembrane domain) of PrrB plays an important role not only in optimally sensing the state of the cbb(3) oxidase but also in maintaining the correct conformation of PrrB, providing optimal autokinase activity.

Base Sequence↗
PubMed2004

Prevalence of congenital heart disease.

BACKGROUND: Today most patients with congenital heart disease survive childhood to be cared for by adult cardiologists. The number of physicians that should be trained to manage these lesions is unknown because we do not know the number of patients. METHODS: To answer this question, the expected numbers of infants with each major type of congenital heart defect born in each 5-year period since 1940 were estimated from birth rates and incidence. The numbers expected to survive with or without treatment were estimated from data on natural history and the results of treatment. Finally, lesions were categorized as simple, moderate, or complex, based on the amount of expertise in management needed for optimal patient care. RESULTS: From 1940 to 2002, about 1 million patients with simple lesions, and half that number each with moderate and complex lesions, were born in the United States. If all were treated, there would be 750,000 survivors with simple lesions, 400,000 with moderate lesions, and 180,000 with complex lesions; in addition, there would be 3,000,000 subjects alive with bicuspid aortic valves. Without treatment, the survival in each group would be 400,000, 220,000, and 30,000, respectively. The actual numbers surviving will be between these 2 sets of estimates. CONCLUSIONS: Survival of patients with congenital heart disease, treated or untreated, is expected to produce large numbers of adults with congenital disease, and it is likely that many more adult cardiologists will need to be trained to manage moderate and complex congenital lesions.

Adult↗
PubMed2004

Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes.

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes--aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis--were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

Adaptation, Physiological↗
PubMed2004

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II.

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.

Bacterial Proteins↗
PubMed2003

Effects of oxygen and light intensity on transcriptome expression in Rhodobacter sphaeroides 2.4.1. Redox active gene expression profile.

The roles of oxygen and light on the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 have been well studied over the past 50 years. More recently, the effects of oxygen and light on gene regulation have been shown to involve the interacting redox chains present in R. sphaeroides under diverse growth conditions, and many of the redox carriers comprising these chains have been well studied. However, the expression patterns of those genes encoding these redox carriers, under aerobic and anaerobic photosynthetic growth, have been less well studied. Here, we provide a transcriptional analysis of many of the genes comprising the photosynthesis lifestyle, including genes corresponding to many of the known regulatory elements controlling the response of this organism to oxygen and light. The observed patterns of gene expression are evaluated and discussed in light of our knowledge of the physiology of R. sphaeroides under aerobic and photosynthetic growth conditions. Finally, this analysis has enabled to us go beyond the traditional patterns of gene expression associated with the photosynthesis lifestyle and to consider, for the first time, the full complement of genes responding to oxygen, and variations in light intensity when growing photosynthetically. The data provided here should be considered as a first step in enabling one to model electron flow in R. sphaeroides 2.4.1.

Gene Expression Profiling↗
PubMed2003

Usefulness of the pediatric electrocardiogram in detecting left ventricular hypertrophy: results from the Prospective Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted HIV Infection (P2C2 HIV) multicenter study.

BACKGROUND: A shortcoming of the pediatric electrocardiogram (ECG) appears to be its inability to accurately detect left ventricular hypertrophy (LVH). This study prospectively assesses the usefulness of the pediatric ECG as a screening modality for LVH. METHODS: Concomitant echocardiograms and ECGs from a large cohort of children who were exposed to the human immunodeficiency virus (HIV; uninfected) and children who were infected with HIV were compared. By use of the values of Davignon et al, qualitative determination of LVH and quantitative criteria for LVH (RV6, SV1, RV6+SV1, QV6, and Q(III) >98% for age, R/SV1 <98% for age, and [-]TV6) were compared to body surface area adjusted for left ventricular (LV) mass z score. Results were then stratified according to weight and weight-for-height z scores. New age-adjusted predicted values were then constructed from children of a mixed race who were HIV-uninfected, < or =6 years old, and similarly assessed. RESULTS: The sensitivity rate was <20% for detecting increased LV mass, irrespective of HIV status; the specificity rate was 88% to 92%. The sensitivity rate of the individual criteria ranged from 0 to 35%; the specificity rate was 76% to 99%. Test sensitivities remained low when stratified by weight and weight-for-height z scores. Areas under the receiver operator characteristic curves were between 0.59 and 0.70, also suggesting poor accuracy of the ECG criteria. By use of new age-adjusted predicted values, the sensitivity rate decreased to <17%, and the specificity rate increased to 94% to 100%. CONCLUSION: The ECG is a poor screening tool for identifying LVH in children. Sensitivity is not improved with revision of current criteria.

Child, Preschool↗
PubMed2003

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides.

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Aerobiosis↗
PubMed2003

Digging deeper: uncovering genetic loci which modulate photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

A new genetic locus was identified in Rhodobacter sphaeroides which is required for optimal synthesis of the light-harvesting spectral complexes as well as for optimal growth under anaerobic conditions with dimethyl sulfoxide (DMSO) as a terminal electron acceptor. The primary structure of the deduced osp gene product shows significant homology to the receiver domain of known response regulators common to bacterial two-component systems. However, site-directed mutagenesis revealed that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway. Paradoxically, the effect of the Osp protein upon spectral complex levels is exerted at the transcriptional level of photosynthesis gene expression. The absence of the Osp protein does not appear to have a general effect on house-keeping metabolism. In cells lacking Osp, the levels of DMSO reductase appear to be normal. The quaternary structure of the Osp protein was determined to be a homodimer and it was directly demonstrated that Osp does not bind to the promoter region of photosynthesis genes as judged by mobility-shift experiments and primary structure analysis.

Amino Acid Sequence↗
PubMed2003

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

Chromosomes, Bacterial↗