Search PubMed⌕ Search

Biomedical subjects

J G Thomas

Publications and source records attributed to J G Thomas.

At least 19 recordsLinked to original sources
PubMed2001

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS).

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.

Biochemistry↗
PubMed2001

An efficient and robust statistical modeling approach to discover differentially expressed genes using genomic expression profiles.

We have developed a statistical regression modeling approach to discover genes that are differentially expressed between two predefined sample groups in DNA microarray experiments. Our model is based on well-defined assumptions, uses rigorous and well-characterized statistical measures, and accounts for the heterogeneity and genomic complexity of the data. In contrast to cluster analysis, which attempts to define groups of genes and/or samples that share common overall expression profiles, our modeling approach uses known sample group membership to focus on expression profiles of individual genes in a sensitive and robust manner. Further, this approach can be used to test statistical hypotheses about gene expression. To demonstrate this methodology, we compared the expression profiles of 11 acute myeloid leukemia (AML) and 27 acute lymphoblastic leukemia (ALL) samples from a previous study (Golub et al. 1999) and found 141 genes differentially expressed between AML and ALL with a 1% significance at the genomic level. Using this modeling approach to compare different sample groups within the AML samples, we identified a group of genes whose expression profiles correlated with that of thrombopoietin and found that genes whose expression associated with AML treatment outcome lie in recurrent chromosomal locations. Our results are compared with those obtained using t-tests or Wilcoxon rank sum statistics.

Acute Disease↗
PubMed2000

ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells.

DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES. However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.

Animals↗
PubMed1999

The double-crush phenomenon--an unusual presentation and literature review.

The double-crush syndrome was initially described by Upton and McComas in 1973. They postulated that nonsymptomatic impairment of axoplasmic flow at more than one site along a nerve might summate to cause a symptomatic neuropathy. This was suggested by their clinical observation that the majority of their patients had a median or ulnar neuropathy associated with evidence of cervicothoracic root lesions. They also hypothesized that one of the constraints on axoplasmic flow could be a metabolic neuropathy, and this is supported by the high association of diabetes and carpal tunnel syndrome. Other researchers have since reported series of patients supporting the frequent association of a proximal and distal nerve compression syndrome, including carpal tunnel syndrome associated with cervical radiculopathy, brachial plexus compression, and diabetic neuropathy. Subsequently, MacKinnon and Dellon have expanded the description of this syndrome to include a) multiple anatomic regions along a peripheral nerve, b) multiple anatomic structures across a peripheral nerve within an anatomic region, c) superimposed on a neuropathy, and d) combinations of the above. We present an unusual case of symptomatic nerve compression caused by two nonanatomic structures within an anatomic region.

Aged↗
PubMed1998

A critical analysis of multiple chemical sensitivities.

Multiple chemical sensitivities (MCS) is a chronic condition of irritation and inflammation of sensory organs, gastrointestinal distress, fatigue, and compromised neurological function, including learning and memory deficits, unpleasant smells, tingling of nerves, and sensory discomfort. Victims report these symptoms after exposure to unfamiliar chemicals. Some studies have linked MCS to immune system dysregulation. MCS is believed to be a disease that spreads between various target organs, and is caused by sensitization to chemicals with very different structures. MCS is often attributed to free radical production and stress, which indirectly cause spreading because of damage to the immune system.

Free Radicals↗
PubMed1998

Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo.

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46 degrees C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50 degrees C. When cultures were allowed to recover from transient exposure to 50 degrees C, all mutations except Deltaibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42 degrees C when combined with the dnaK756 or groES30 alleles, while the Deltaibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46 degreesC compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.

Adenosine Triphosphatases↗
PubMed1998

Long-term sub-antimicrobial doxycycline (Periostat) as adjunctive management in adult periodontitis: effects on subgingival bacterial population dynamics.

Previous trials had indicated that various schedules of sub-antimicrobial doxycycline significantly reduced gingival crevicular fluid (GCF) collagenase activity in adult patients with periodontitis with no evidence of emergent tetracycline-resistant (Tcr) marker oral flora. The purpose of this nine-month study was to expand these observations, emphasizing newer microbial diagnostic methods. Subgingival paper point samples were obtained at baseline (BL), 3, 6, and 9 months. Four subject treatment groups in a double-blind design were evaluated by mechanical scaling and root planing (SRP) and/or 20 mg doxycycline BID (Periostat). Thirty-eight patients entered the study at baseline (BL). Dark-field microscopy on 260 samples showed that morphotype distribution was independent of treatment schedule. Culture analysis of the 3 most prevalent isolates recovered showed that Streptococcus and Prevotella species accounted for approximately 85% of the 724 cultures. There did not appear to be any overgrowth or replacement by opportunistic oral flora. Of 658 susceptibility patterns evaluated by Etest, the MIC50/90 and mode MIC showed stable patterns, independent of treatment group. Our findings were different from those of previously published reports, but may be partly explained by the lack of universally standardized methods in oral microbiology and interpretive criteria for susceptibility testing.

Adult↗
PubMed1997

Rapid diagnosis of acute eosinophilic pneumonia (AEP) in a patient with respiratory failure using bronchoalveolar lavage (BAL) with calcofluor white (CW) staining.

A diagnosis of exclusion, acute eosinophilic pneumonia (AEP) is an acute febrile illness with respiratory impairment, diffuse pulmonary infiltrates, and bronchoalveolar lavage (BAL) fluid eosinophilia. Whether pulmonary eosinophilia in AEP is primary or secondary remains undetermined. We report here a 22-year-old auto mechanic with severe AEP and acute respiratory failure who required intubation and ventilatory support. The patient's bronchoalveolar lavage (BAL) fluid was analyzed using cultures, cytology, Wright/Giemsa, Gram, Gomori-methenamine-silver (GMS), and calcofluor white (CW) stains (1). Despite extensive evaluation, no infectious etiology was found. CW staining helped us rapidly to exclude Pneumocystis carinii or fungal infection and to focus attention toward the diagnosis of AEP. Transbronchial biopsy was unnecessary and supportive therapy without systemic glucocorticoids was followed by recovery within a few weeks. In this case, bronchoalveolar lavage with CW staining was of great assistance in the rapid diagnosis and initial management of AEP. Our literature review found no prior article using CW staining for evaluation of AEP.

Acute Disease↗
PubMed1997

Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli.

The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished. Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'-beta-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.

Cloning, Molecular↗
PubMed1997

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. To fold or to refold.

The high-level expression of recombinant gene products in the gram-negative bacterium Escherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as the cis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.

Bacterial Proteins↗
PubMed1997

Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli.

Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

Amino Acid Sequence↗
PubMed1996

Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins.

PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.

Chaperonin 10↗
PubMed1996

Laboratory implementation of a rapid three-stain technique for detection of microorganisms from lower respiratory specimens.

A rapid, cost-effective method for the evaluation of lower respiratory specimen has become increasingly important in the diagnosis of pulmonary diseases in immunocompromised patients. In the past, the technically demanding, time-consuming, and expensive Gomori-methenamine-silver (GMS) stain was the principal means for the evaluation of these specimens. In this study, we compared the GMS stain with a new rapid, three-stain protocol for the evaluation of lower respiratory specimens. Lower respiratory specimens were obtained by bronchoalveolar lavage (BAL). Conventional Wright/Giemsa and Gram stains were utilized, as well as a contemporary strain, calcofluor white (CW). A cell count was performed on the BAL specimens, and cytospins were stained by the three stains. The calcofluor white-stained slides were examined with an epi-fluorescent microscope, whereas the other stains were evaluated with a conventional light microscope. Gomorimethenamine-silver (GMS), acid-fast bacillus (AFB), and Papanicolaou (PAP) stains were performed as controls. Thirty-two BAL procedures were performed in 20 (63%) male patients and 12 (37%) female patients. The clinical diagnosis was pneumonia in 31% of the patients, malignant hematologic disease in 28%, acute respiratory distress syndrome (ARDS) in 9%, and acquired immunodeficiency syndrome (AIDS) in 28%. Of these specimens, 78% were adequate for interpretation and 22% were inadequate. Bacteria were found in 50% (16/32) of all BALs, fungi were found in 9% (3/32), and Pneumocystis carinii was found in 9% (3/32). Gram-positive bacteria were most frequently found in patients with pneumonia (80%, 4/5), whereas P. carinii was identified in patients with AIDS. There were no false-positive results. One CW stain was equivocal for P. carinii due to high fluorescent background. Laboratory implementation of the rapid, three-staining technique was accomplished without difficulty in microbiology and hematology laboratory sections. Specimen evaluation with the rapid staining protocol was technically easy to perform; however, experience in ultraviolet fluorescent microscopy was crucial for interpretation of CW stain. All results were available in 2 hr, cost was reduced by 30%, and the assays were available 7 days/week. Further studies are ongoing to substantiate the sensitivity, specificity, and predictive value of this technique, as well as clinical guidelines for its optimal utilization.

Adult↗
PubMed1996

Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines.

We have systematically investigated the influence of mutations in the sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines on the folding of preS2-beta-galactosidase. This 120 kDa fusion protein between the hepatitis B surface antigen preS2 sequence and beta-galactosidase was synthesized in a highly soluble and enzymatically active form in wild-type Escherichia coli cells cultured at temperatures between 30 degrees C and 42 degrees C, but aggregated extensively in an rpoH165 (Am) mutant. Proper folding was partially restored upon co-overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced. The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40-60% lower relative to a dnaK756 mutant or isogenic wild-type cells at 30 degrees C and 37 degrees C. At 42 degrees C, only 10-40% of the wild-type activity was present in each of the early-folding-factor mutants. Although the synthesis levels of preS2-beta-galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37 degrees C or 42 degrees C. By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2-beta-galactosidase at temperatures up to 42 degrees C. Our results are discussed in terms of the current understanding of the E. coli protein-folding cascade. The potential usefulness of heat-shock protein mutants for the production of soluble proteins in an inclusion-body form is addressed.

Escherichia coli↗
PubMed1996

The early parenting of twins.

The anticipatory guidance for the parents of twins involves information on feeding, the parent-twin relationship, and promoting a sense of self. Parents optimally attach to one infant only, a concept known as monotropy. Ambivalence, anxiety, and unequal attachments may result from the stressful nature of raising twins. The early diagnosis of twins, visualization via ultrasound, and postpartum identification of the distinctive aspects of each infant help establish separate bonds. This individualization promotes a sense of self, self-esteem, and enhances individuation, the process by which the personality is differentiated, developed, and expressed. The twin situation is triadic; the infants develop attachments to the primary care giver and their co-twin. Problems with this complex association include delayed onset of speech, decreased speech production, and cryptophasia (secret language) between twins. Strategies to prevent speech delay include as much one-on-one time as possible and consistent intervention and correction of speech errors. Anticipatory guidance seeks to identify potential sources of stress with the goal of seeking out advice, accepting any assistance offered, and attending to twins at the desired level of individualization.

Child Development↗