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Biomedical subjects

J Wayne

Publications and source records attributed to J Wayne.

At least 19 recordsLinked to original sources
PubMed2006

A fluorescence polarization assay for inhibitors of Hsp90.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.

Adenosine Triphosphatases↗
PubMed2006

Re-engineering the target specificity of Clostridial neurotoxins - a route to novel therapeutics.

The ability to chemically couple proteins to LH(N)-fragments of clostridial neurotoxins and create novel molecules with selectivity for cells other than the natural target cell of the native neurotoxin is well established. Such molecules are able to inhibit exocytosis in the target cell and have the potential to be therapeutically beneficial where secretion from a particular cell plays a causative role in a disease or medical condition. To date, these molecules have been produced by chemical coupling of the LH(N)-fragment and the targeting ligand. This is, however, not a suitable basis for producing pharmaceutical agents as the products are ill defined, difficult to control and heterogeneous. Also, the molecules described to date have targeted neuroendocrine cells that are susceptible to native neurotoxins, and therefore the benefit of creating a molecule with a novel targeting domain has been limited. In this paper, the production of a fully recombinant fusion protein from a recombinant gene encoding both the LH(N)-domain of a clostridial neurotoxin and a specific targeting domain is described, together with the ability of such recombinant fusion proteins to inhibit secretion from non-neuronal target cells. Specifically, a novel protein consisting of the LH(N)-domains of botulinum neurotoxin type C and epidermal growth factor (EGF) that is able to inhibit secretion of mucus from epithelial cells is reported. Such a molecule has the potential to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease.

Botulinum Toxins↗
PubMed2001

Biomechanical comparison of ankle arthrodesis techniques: crossed screws vs. blade plate.

Many different techniques for ankle arthrodesis have been described. Experience at our institution with crossed screws internal fixation has not met the 90+% union rate reported in the literature. A compression blade plate is one technique for ankle arthrodesis which has not been evaluated biomechanically. A biomechanical study comparing two groups of sawbone ankle fusion constructs fixed with crossed screws and compression blade plates was performed in order to evaluate the stiffness and rigidity of these two arthrodesis techniques. The crossed screws construct demonstrated superior stiffness during dorsiflexion (p < 0.001) and valgus (p < 0.001) loading. The two constructs were found to have equal strength in resisting plantarflexion, varus and torsional loads although there was a trend for greater resistance by the crossed screws construct. These findings lend biomechanical support to the use of crossed screws for tibiotalar arthrodesis.

Ankle Joint↗
PubMed2000

Mechanical comparison of fixation techniques for the tibial tubercle osteotomy.

Tibial tubercle osteotomies currently are used as an exposure technique for revision total knee arthroplasty and for distal patellofemoral realignment. A review of the literature reveals no biomechanical studies that evaluate methods of osteotomy fixation in terms of static strength. This study evaluates the fixation strength of common techniques used to repair tibial tubercle osteotomies. Bevel and stepcut tibial tubercle osteotomies were created in 36 anatomic specimen knees and were repaired with either two 4.5-mm cortical screws or 18-gauge stainless steel cerclage wire. The failure load for the bevelcut osteotomies repaired with two-screws was 1,654 +/- 359 N; for the bevelcut osteotomies repaired with three cerclage wires, 622 +/- 283 N; for the stepcut osteotomies repaired with three cerclage wires, was 984 +/- 441 N; and for the stepcut osteotomy repaired with four cerclage wires, 1,099 +/- 632 N. This study shows that two bicortical screws provide the greatest static fixation strength for repairing tibial tubercle osteotomies. When repairing tibial tubercle osteotomies for distal patellofemoral realignment, screw fixation would provide the most reliable fixation. However, the placement of screws around the stem of a revision arthroplasty tibial component is difficult. Cerclage wires are easier to place and provide solid static fixation, especially with the addition of a proximal stepcut osteotomy.

Aged↗
PubMed1999

Bcl-2-induced changes in E2F regulatory complexes reveal the potential for integrated cell cycle and cell death functions.

Proliferation and cell death are tightly linked fates during cell and tissue differentiation. In the past few years, it has been shown that Bcl-2 exhibits a potent cell cycle inhibitory effect, in addition to its better known role in the antagonism of cell death. In the present study, we show that the cell cycle effects of Bcl-2 apparently occur at the level of E2F control of gene transcription. Under conditions of normal cell growth, or under conditions that lead to cell death in the absence of Bcl-2, bcl-2 expression results in a reduction of free (active) E2F isoforms and in an increase in the formation of higher-order (inactive) complexes. Bcl-2-induced changes in E2F complex formation are paralleled by an apparent increase in pRb regulatory activity, by the up-regulation of p130 protein expression, and by the formation of E2F/p130 complexes at the expense of those consisting of E2F/p107. Cells lacking bcl-2 expression respond to growth factor withdrawal in the opposite manner, by the liberation of E2F from inactivating complexes and by continued cell cycle leading to cell death. These analyses reveal a mechanism for cell cycle regulation by Bcl-2 that occurs at the level of E2F transcriptional activity. Further, since specific E2F activities are clearly linked to the induction of cell death, these findings may help to consolidate the cell survival and cell cycle effects of Bcl-2 through a common transcriptional mechanism.

Carrier Proteins↗
PubMed1998

A new look at an old option in the treatment of early-stage prostate cancer: hormone therapy as an alternative to watchful waiting.

Watchful waiting is an attractive option in the management of early-stage, low-grade prostate cancer because of the high financial costs and complication risks associated with surgery and radiotherapy. Despite the drawbacks of current local therapy, neither treatment can demonstrate a discernible survival benefit over observation alone. Even the slowest progressing disease, however, can potentially develop into a deadly medical problem. As a result, physicians and patients frequently have difficulty accepting untreated cancer. Therefore, we propose that another option be considered in cases of prostate cancer that would otherwise qualify for observation alone: the use of two oral hormonal agents, flutamide and finasteride, to achieve complete androgen blockade. Some evidence exists which suggests that such therapy may improve symptom-free survival, and perhaps overall survival as well. This 'aggressive' form of 'conservative' therapy may satisfy patient concerns that are not adequately addressed by current forms of therapy.

5-alpha Reductase Inhibitors↗
PubMed1998

Comparison of two proximal osteotomies for the treatment of hallux valgus.

Moderate to severe deformities of hallux valgus can be corrected with combination procedures, such as a proximal crescentic metatarsal osteotomy and a distal soft tissue procedure. Because crescentic osteotomy allows for motion in all planes, inadvertent metatarsal elevation can result in metatarsalgia. The crescentic shelf osteotomy (CSO) provides a plantar shelf, decreasing the complication of dorsiflexion fixation. Eighteen polyurethane foam specimens in three groups were prepared and tested to failure on a servohydraulic Instron testing machine. The mechanical characteristics of stiffness, deformation, ultimate failure load, and stored energy were compared between single-screw fixation crescentic osteotomies and single- and dual-screw CSOs in molded polyurethane foam metatarsal sawbones. In addition, 12 cadaver specimens were randomly divided, and a CSO or crescentic osteotomy was performed. Preosteotomy and postosteotomy intermetatarsal, dorsiflexion, and pronation angles were compared from radiograph measurements. The results showed comparable mechanical characteristics among the groups, as measured by the area under the curve (P=0.95), ultimate failure load (P=0.35), deformation (P=0.63), and stiffness (P=0.21). Greater improvements were seen in the CSO group compared with the crescentic osteotomy group in correction of the intermetatarsal angle (4.8 degrees compared with 3.2 degrees) and of the first metatarsal plantarflexion (2.3 degrees compared with 3.2 degrees of dorsiflexion). However, these differences were insignificant (P=0.10 and P=0.41) with the numbers available. Compared with the crescentic osteotomy, a CSO may possibly provide easier initial fixation but similar mechanical properties.

Bone Screws↗
PubMed1997

The Tsp45I restriction-modification system is plasmid-borne within its thermophilic host.

Thermus species YS45 harbors two small cryptic plasmids of 5.8 (pTsp45s) and approximately 12 kb (pTsp45I). Plasmid pTsp45s has been entirely sequenced, revealing three significant ORFs. In addition to a previously reported thermophilic plasmid-encoded replication protein (Rep), pTsp45s contains two genes for the Tsp45I methyltransferase (M.Tsp45I) and restriction endonuclease (Tsp45I). These two converging genes (tsp45IM and tsp45IR) overlap by 4 bp at their stop codons within an XbaI site. M.Tsp45I (413 aa, 47.0 kDa, recognizing 5'-GTSAC-3') is highly homologous to other m6A-methyltransferases, especially M.EcaI (recognizing 5'-GGTNACC-3'). Tsp45I (332 aa, 37.4 kDa, cleaving 5'-/GTSAC-3') is not homologous to M.Tsp45I, or to other restriction endonucleases. Recombinant Tsp45I is stably produced in E. coli, and cleaves DNA at 65 degrees C with the same specificity as the native enzyme. Therefore, the thermophilic Tsp45I restriction-modification system is plasmid-borne within its native host.

Amino Acid Sequence↗
PubMed1997

Identification of a thermophilic plasmid origin and its cloning within a new Thermus-E. coli shuttle vector.

A pUC19-based vector has been generated for selecting functional thermophilic origins (oris) of Thermus ssp. Once combined with thermophilic DNA, the vector can be amplified in ampicillin resistant (Ap(R)) E. coli, prior to transformation and kanamycin (Km) selection in Thermus thermophilus. The Km(R) Thermus transformants replicate any newly-formed shuttle vectors via introduced thermophilic oris. Using this "ori-selecting" vector, three novel thermophilic oris were cloned from randomly digested Thermus cryptic plasmid DNA. These shuttle vectors are useful for genetic analyses, as well as protein engineering within thermophiles. The smallest ori-containing sequence of 4.2 kb has been subcloned, sequenced, and further refined to 2.3 kb. A significant ORF of 341 amino acids (aa), with a Thermus promoter and RBS, is found within the thermophilic ori. Deleting part of this ORF abolishes the shuttle vector's ability to replicate in T. thermophilus. Therefore, we postulate that this ORF encodes a replication protein (Rep) necessary for thermophilic plasmid replication. The thermophilic ori also contains two sequences which resemble DnaA boxes.

Amino Acid Sequence↗
PubMed1997

The presence of K-12 ras mutations in duodenal adenocarcinomas and the absence of ras mutations in other small bowel adenocarcinomas and carcinoid tumors.

BACKGROUND: Adenocarcinomas and carcinoid tumors are the most common malignant tumors of the small intestine. K-ras oncogene mutations at codon 12 are common in gastric, pancreatic, and colon carcinomas, with an incidence of 35-88%. K-ras mutations have not been extensively studied in either adenocarcinomas or carcinoid tumors of the small bowel. The purpose of this study was to determine whether ras mutations play an important role in the formation of these tumors. METHODS: Archival tissues from 28 adenocarcinomas and 22 carcinoid tumors of the small bowel were studied, along with archival tissues from 32 adenocarcinomas of the large bowel, which were used as controls. DNA from the small intestine tumors was analyzed for K-ras, H-ras, and N-ras oncogene mutations at codons 12, 13, and 61, using polymerase chain reaction and sequence specific oligonucleotide hybridization techniques. Large bowel adenocarcinomas were analyzed for K-ras mutations at codons 12 and 13. RESULTS: A point mutation of K-ras at codon 12 was detected in 4 of 28 (14.3%) of the small bowel adenocarcinomas, in 12 of 32 (37.5%) of the large bowel adenocarcinomas, and in 0 of 22 small intestine carcinoid tumors. No other K-ras, H-ras, or N-ras mutations were detected in any of the small bowel tumors. Each small intestine K-ras mutation was found in a duodenal adenocarcinoma (4 of 12 cases, 33%), whereas none occurred in 16 other jejunal or ileal adenocarcinomas. CONCLUSIONS: K-ras mutations appear to play a significant role in the pathogenesis of duodenal adenocarcinomas, but they do not appear to be important in the development of jejunal or ileal adenocarcinomas or of carcinoid tumors of the small intestine.

Adenocarcinoma↗
PubMed1996

The effects of mental health factors on ambulatory care visits by rural teens.

This study sought to determine the effects of mental health variables on rural adolescents' use of ambulatory health care services and whether these effects varied across common outpatient settings. Using a cross-sectional survey design, 2,297 adolescents who attended public schools in grades 7 through 12 in one isolated rural Mississippi River Delta county were assessed via a standardized health behavior survey. This self-report measure inquired about relevant health behaviors such as alcohol use, depressive symptoms, and health service use. The students' mean age was 15 years and 58 percent of the sample were black. Approximately 11 percent of the sample reported symptoms of depression, 16.5 percent reported problem drinking, and slightly fewer than 6 percent reported both. After controlling for predisposing, enabling, and need factors, the reporting of depressive symptoms, problem drinking, or both was related to an increased number of outpatient visits in three of four sites examined. However, differences among sites were observed. These data suggest that mental health problems are associated with increased visits to ambulatory settings, and these problems affect service use differentially. Thus, effective interventions and better linkages between ambulatory settings and mental health providers may reduce unnecessary use.

Adolescent↗
PubMed1996

Contribution of the deltoid ligament to ankle joint contact characteristics: a cadaver study.

Changes in ankle biomechanics lead to altered load transmission through the ankle joint, possibly predisposing it to osteoarthritis. Contributions of the different bands of the deltoid ligament to the contact characteristics in the ankle were examined. Fifteen normal cadaveric lower extremities were axially loaded to 445 N after intra-articular Fuji film placement. Ankles were tested in neutral, 10 degrees dorsiflexion, and 10 degrees plantarflexion. Repeated testing was done following sequential sectioning of the deltoid ligament, and the contact characteristics were analyzed. The greatest significant tibiotalar changes (P < 0.0001) occurred after sectioning of the tibiocalcaneal fibers of the superficial deltoid ligament complex. Contact areas decreased up to 43%, peak pressures increased up to 30%, and centroids moved 4 mm laterally, on average. In contrast, sectioning of the other bands led to insignificant changes in joint contact characteristics. The data indicate that significant changes in contact characteristics occur before radiographic evidence of deltoid ligament damage is evident, and may indicate that greater attention to the medial side of the ankle is indicated to restore normal biomechanics to this joint.

Analysis of Variance↗
PubMed1994

Transformation by homeobox genes can be mediated by selective transcriptional repression.

Altered transcription is a recurrent theme in the field of cancer biology. But despite the central role of transcription in transformation, little is known about the mechanism by which dominant nuclear oncogenes induce malignancies. Homeobox family proteins are prominent examples of transcriptional regulators which control development and can function as oncogenes. Here we explore the molecular basis for transformation by this class of regulators using Oct-2 and Oct-1. We show that the DNA binding POU domains of these proteins are selective and sequence-specific transcriptional repressors that produce malignant lymphomas when they are expressed in T cells of transgenic mice. Mutagenesis experiments identified a specific set of promoters, those containing octamer regulatory elements, as the targets for transformation by selective inhibition of gene expression.

Animals↗
PubMed1994

TCR selection and allelic exclusion in RAG transgenic mice that exhibit abnormal T cell localization in lymph nodes and lymphatics.

RAG-1 and RAG-2 are developmentally regulated genes that are essential for V(D)J recombination and lymphocyte development. Expression of RAG-1 and RAG-2 by thymocytes is normally limited to cells that have not completed selection. We have previously documented that persistent expression of the recombinase activating genes (RAG) in transgenic mice results in aberrant thymic development, altered lymphatic microanatomy, and a profound immunodeficiency. Here we further document the pathologic changes found in TG.RAG-1,2 mice and examine the role of TCR recombination and positive and negative thymic selection, as well as allelic exclusion, in the etiology of the phenotype. We find that neither selection nor TCR allelic exclusion can be overcome by transgenic expression RAG-1 and RAG-2 under the control of an lck promoter.

Alleles↗
PubMed1994

A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development.

RAG-1 and RAG-2 are developmentally regulated genes that are essential for the assembly of antigen receptors in lymphoid cells. Here we describe transgenic mice that carry RAG-1 and RAG-2 under the control of the proximal lck promoter. Persistent expression of RAG-1 and RAG-2 was associated with incomplete thymopoiesis and profoundly compromised cellular immunity. In addition, RAG transgenic mice rapidly developed lymphadenopathy, splenomegaly, and lymphocytic perivascular infiltrates. These effects required both RAG-1 and RAG-2, since mice that carried either gene exclusively were indistinguishable from wild-type controls. We propose that in addition to a previously documented role in V(D)J recombination, RAG-1 and RAG-2 expression must be properly regulated for completion of normal T cell development

Animals↗
PubMed1993

Continuing trends in the prevalence of right-sided lesions among colorectal carcinomas.

The shift of colorectal carcinoma location toward the proximal colon has been reported. This study documents that this statistically significant trend has continued through 1992. An increase in transverse and descending colon cancers is now apparent also. Only 59% of all large-bowel cancers occurred distal to the descending colon between 1978 and 1992. Both right-sided and distal large-bowel cancers have significantly decreased in size, yet the incidence and frequency of lymph node metastases have not changed over a 65-year interval (from 1928 to 1992). This constant proportion of lymph node metastases may suggest distinct biological subsets of cancers (lymph node avid vs lymph node avoidance). The progression from small size with fewer metastases to large size with more lymph node metastases occurs only in some of the smallest distal colorectal cancers.

Boston↗