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Biomedical subjects

R M Patterson

Publications and source records attributed to R M Patterson.

At least 19 recordsLinked to original sources
PubMed1997

Load mechanics of the wrist.

During the last decade, we have been studying load mechanics under a variety of conditions using pressure-sensitive film and, more recently, three-dimensional reconstructions and motion analysis. In all of these studies using pressure-sensitive film, the simulated pathologic or traumatic conditions that were tested showed that all areas in which an increase in contact area or pressure occurred, localized to one area of one joint, coincided with areas in which degenerative changes occurred in the simulated clinical situation. The areas in which there was a decrease or no change in contact area or pressure coincided with areas that were spared from degenerative changes in simulated clinical situations. More recent work has demonstrated that normal carpal kinematics during wrist flexion and extension do not have an instantaneous screw axis that is fixed in or limited to the capitate. These findings change the understanding previously based on studies suggesting that the center of rotation was fixed in the capitate. It was noted that during global wrist motion the radiolunate joint contributes more motion and flexion than the capitolunate joint, but the capitolunate joint contributes more motion and extension than the radiolunate joint. It also was demonstrated that translational motion is a normal component of carpal kinematics.

Biomechanical Phenomena↗
PubMed1997

Normal wrist kinematics and the analysis of the effect of various dynamic external fixators for treatment of distal radius fractures.

The purpose of this study was to evaluate the effect that external fixation devices used for distal radius fractures have on normal carpal kinematics, using high speed video data acquisition. Cadaver forearms were stabilized, allowing free excursion of the wrist for passive range of motion. Synergistic muscle tension was simulated by looping the extensor and flexor pairs of the wrist. Global wrist flexion and extension, intercarpal angles, and the instantaneous screw axis (ISA) were studied. These parameters allowed a quantification of normal carpal kinematics and comparison to the kinematics of the carpal bones with external fixators attached. This study shows that normal carpal kinematics during wrist flexion and extension do not have an ISA fixed in or limited to the capitate. In addition, the ISA data provide evidence that translational motion is a real and measurable component of normal carpal motion. This would change the understanding of carpal kinematics in previous studies that suggested that the center of rotation was fixed in the capitate. The proximal carpal (radiolunate) joint contributes more motion (has a greater rotation angle) in normal global wrist flexion and the midcarpal (capitolunate) joint contributes more motion in normal global wrist extension. In addition, the global range of motion and intercarpal joint angle of the wrist are limited with the application of any of the external fixators tested. None of the external fixators allowed normal ISA vector surfaces during wrist motion.

Adult↗
PubMed1996

HSP binding and mitochondrial localization of p53 protein in human HT1080 and mouse C3H10T1/2 cell lines.

In normal cells, the tumor suppressor actions of p53 protein are mediated by specific DNA binding and protein-protein interactions within the nucleus. Mutant p53 proteins, however, often assume an aberrant conformation devoid of tumor suppressor activity and newly capable of binding to the cognate or inducible HSP70. Recent reports from our laboratory and others show that additional unknown proteins may also complex with mutant p53. In this study, we characterize p53:HSP complexes and their subcellular location in the transformed cell lines, human HT1080 and murine C3H10T1/2, which both contain aberrant p53 conformers. Immunoprecipitation and SDS-PAGE of p53 from whole cell lysates revealed the additional presence of a broad 70 kDa band and a 90 kDa band in both lines, while p53 isolated from nuclear lysates was free from other proteins. 2D-PAGE was used to isolate and identify HSP members from cytoplasmic and nuclear lysates by immunoprecipitation, Western blotting and protein sequencing. Anti-p53 immune complexes from cytoplasmic lysates contained not only HSC70 but also GRP75, GRP78 and a weakly basic 90 kDa protein, which may be related to HSP90. The inducible form of HSP70 was not complexed to p53 protein, even though expressed in these cells. Analysis of anti-HSP70, anti-GRP75 and anti-HSP90 immune complexes suggests that HSP members exist as performed complexes in the cytoplasm, but not the nucleus. The presence of the mitochondrial and endoplasmic reticular chaperones, GRP75 and GRP78, in p53:HSP complexes suggested that p53 might be found in these cytoplasmic organelles which was confirmed in mitochondria by biochemical and immunoelectron microscopic evidence. These studies suggest that newly identified members of p53:HSP complexes represent components of a chaperone program which affects the subcellular distribution of p53 protein in these transformed lines.

Amino Acid Sequence↗
PubMed1996

Human p53 expressed in baculovirus-infected Sf9 cells displays a two-dimensional isoform pattern identical to wild-type p53 from human cells.

Baculovirus expression of human p53 protein, a nuclear cell cycle regulator, was examined in Sf9 cells and compared to native p53 synthesized in primary human cells. Maximum expression of the recombinant p53 protein occurred 48 h postinfection. De novo synthesis of the protein was evident for only 2 days postinfection; however, in pulse-chase studies, 30% of the synthesized protein remained stable up to 5 days. Seventy-seven percent of immunoprecipitated, [35S]-methionine-labeled, recombinant p53 protein resided in the cytoplasm of Sf9 cells, while 15% localized to the nucleus and 8% was released extracellularly. Separation of modified p53 protein, by charge and molecular weight, was accomplished by two-dimensional PAGE, and the electrophoretic pattern of the recombinant protein was identical to the wild-type protein from primary human mammary epithelial cells, indicating that the posttranslational modifications of the recombinant protein in this system are similar to those in primary human cells. Eleven isoforms focused between pI 5.75 and pI 6.5. The recombinant p53 isoforms were phosphorylated by 32P-labeling. Phosphatase digestion of immunoprecipitated p53 effectively removed phosphorous groups from the recombinant protein, reducing the number of isoforms from 11 to 2, demonstrating that phosphorylation is the major posttranslational event in the recombinant protein.

Animals↗
PubMed1996

Tumor suppressor p53 gene forms multiple isoforms: evidence for single locus origin and cytoplasmic complex formation with heat shock proteins.

The tumor suppressor protein p53 is a major cell cycle control factor, and mutations in p53 are the most common genetic lesion found in human tumors, resulting in loss of function and contributing to malignant transformation. This report reviews several studies which show that p53 protein appears as at least eleven isoforms having the same amino acid backbone but varying in charge by level of phosphorylation. All isoforms are derived from a single locus, which indicates that p53 activity is modulated by post-translational modification. In addition, mutant p53 forms hetero-oligomers with two families of proteins: HSP70 and a 90 kDa group similar to HSP90. Cytoplasmic complexes are most likely formed to protect p53 from proteolysis and are probably involved in translocation of activated p53 from the cytoplasm to the nucleus for transactivation of other cell cycle control genes.

Animals↗
PubMed1995

Extrinsic wrist ligaments in the pathomechanics of ulnar translation instability.

A description of the various combinations of ligament disruptions necessary for ulnar translation instability is presented based on a series of cadaver dissections and load studies. Dissections and load studies demonstrated that major ligament disruption, even to the extent that only the dorsal ulnolunate, the palmar ulnolunate or the radioscaphocapitate, and the long radiolunate ligaments were left intact, failed to result in meaningful ulnar translation of the carpus. The radioscaphocapitate ligament alone could not prevent significant ulnar translation. Palmar translation instability was evident with less ligament disruption than that needed for ulnar translation and was always evident if there was ulnar translation. These studies imply that ulnar translation represents a much more global ligament disruption and instability than previously suggested in the literature.

Aged↗
PubMed1995

Carpal bone anatomy measured by computer analysis of three-dimensional reconstructions of computed tomography images.

Using quantitative analysis of three-dimensional reconstructions of computed tomography scan data, a normative database of carpal bone morphology was built. Thirty-five wrists were imaged in a computed tomography scanner. Each slice was processed to determine the bone edges and assembled as a three-dimensional model by stacking. Quantitative measurements of volume, surface area, maximum length, and intercarpal distances were then assessed. A reliable three-dimensional carpal height ratio was calculated by dividing the carpal height (minimum distance between the fourth metacarpal and the radius) by the capitate maximum length. For volume, maximum length, and surface area, the order for the eight carpal bones with respect to size (in descending order) were: capitate, hamate, scaphoid, trapezium, lunate, trapezoid, triquetrum, and pisiform. Male wrists were significantly larger than female wrists. There were no significant differences in the relative dimensions between left and right wrists, or between left and right wrists of matched pairs. This technology offers automated analysis of three-dimensional geometric carpal information and the opportunity to obtain a body of information about normal and abnormal morphology as well as spatial relationships between carpal bones.

Adolescent↗
PubMed1995

Phosphor image analysis of human p53 protein isoforms.

Phosphor imaging was evaluated for detection, quantitation and resolution of multiphosphorylated protein isoforms separated by two-dimensional gel electrophoresis. A nuclear phosphoprotein, p53, was isolated by immunoprecipitation after biosynthetic labeling with 35S, 32P or 33P in cultured human cells. Of the three radionuclides, 35S was the most sensitive in detection after a 1-week exposure, although shorter exposure times were effective. In dividing cells, 11 35S-labeled isoforms were found, of which 10 were phosphorylated by 33P and 32P. Exposure of phosphonuclides for one half-life showed that 33P radiolabeling produced better resolution among isoforms than 32P but was less sensitive in detection. Volume integration showed phosphorylated isoforms comprised from 1% to 25% of total isoform signal. The relative phosphorylation of each p53 isoform was estimated by normalizing 33P or 32P isoform volumes with the corresponding 35S volume and showed progressive phosphorylation of acidic isoforms. Additionally, phosphor imaging capably detected quantitative changes among individual isoforms after experimental modulation of the isoform pattern by serum deprivation. The described electrophoretic isolation and quantitation procedures should find general application in discerning active and inactive phosphoisoforms for eventual identification.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Altered protein synthesis in p53 null and hemizygous transgenic mouse embryonic fibroblasts.

Embryonic fibroblasts derived from p53-deficient transgenic mice showed distinct phenotypic and biological changes in vitro. In this study, we investigated the possible impact of p53 on the synthesis of other cellular proteins by comparing the protein profiles of p53 null (-/-), hemizygous (+/-) and p53 positive homozygous (+/+) cells using high resolution two dimensional gel electrophoresis. A total of more than 850 proteins were detected in each cell line labeled with 35S-methionine by using computerized image analysis, and a number of proteins were detected with qualitative or quantitative changes in p53-/- cells and to a lesser extent in p53+/- cells. Specifically, seven proteins became undetectable, and no new proteins were detected in p53-/- cells. Neither newly expressed nor absent proteins were detected in p53+/- cell line. Quantitatively, a total of 97 and 59 proteins were detected with significant quantitative changes (3 fold or greater) in p53-/- and p53+/- cells, respectively. Generally, most protein changes fell into one of the following four patterns: 1) progressively decreased synthesis in cells from p53+/+ to p53+/- to p53-/- cells; 2) progressively increased synthesis in cells from p53+/+ to p53+/- to p53-/- cells; 3) decreased synthesis only in p53-/- cells; and 4) increased synthesis only in p53-/- cells. A 70 kD heat shock protein (Hsp 70) was identified and showed a greater than 1,000-fold increase in p53-/- cells compared to that in p53+/+ cells. Transferrin, tropomyosin, and proliferating cell nuclear antigen (PCNA) have also been identified and measured in this study. Synthesis of transferrin and tropomyosin was significantly increased or decreased, respectively in p53-/- cells, whereas expression of PCNA showed no significant change in p53-/- cells despite their much higher (3-4 times) proliferation rate than the other two cell lines (p53+/+ and p53+/- cells). We conclude that disruption of a single important gene, p53, results in a cascade of protein changes which are related to the loss of p53 mediated negative growth effects on cell cycle control.

Alleles↗
PubMed1994

Deletion 5q35.3.

We report on a 15-month-old boy with a de novo deletion of the terminal band of 5q, macrocephaly, mild retrognathia, anteverted nares with low flat nasal bridge, telecanthus, minor earlobe anomalies, bell-shaped chest, diastasis recti, short fingers, and mild developmental delay.

Abnormalities, Multiple↗
PubMed1994

Phenotypic change and altered protein expression in X-ray and methylcholanthrene-transformed C3H10T1/2 fibroblasts.

The morphology, growth properties and cellular protein patterns from parent and two transformed C3H10T1/2 cell lines were analyzed to associate the phenotypic and protein differences with cell transformation. Transformed 10T1/2 cells were obtained by colony isolation after exposure of parent 10T1/2 cells to methylcholanthrene (MCA-1 cell line) or X-ray irradiation (XR-III cell line). Compared to parent 10T1/2 and MCA-1 cells, XR-III cells were much smaller in size and exhibited the highest growth rate, greatest cell saturation density, increased plating efficiency and greater expression of proliferating cell nuclear antigen. MCA-1 cells showed intermediate characteristics between parent and XR-III cells. Among the three cell lines, only XR-III cells showed anchorage-independent growth in soft agar. When [35S]methionine-labeled whole cell lysate proteins were separated by two-dimensional polyacrylamide gel electrophoresis, computer comparison algorithms revealed a 97% similarity in protein profiles among almost 800 proteins detected from each cell line. However, comparison of proteins patterns of the transformed cell lines to that of parent 10T1/2 cells showed that 30 and 20 proteins were induced or repressed in XR-III cells and MCA-1 cells, respectively. Similarly, 81 and 24 proteins showed significant quantitative changes (threefold or greater) in XR-III and MCA-1 cells, respectively, as compared with parent 10T1/2 cell proteins. The anchorage-independent growth and increased proliferation properties of XR-III cells suggest a later stage of transformation compared to MCA-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Agar↗
PubMed1994

Separation and sequencing of familiar and novel murine proteins using preparative two-dimensional gel electrophoresis.

Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium-binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mot-1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purification method for protein isolation and characterization from an important in vitro murine model for the study of carcinogenesis.

Amino Acid Sequence↗
PubMed1993

DNA confirmation of congenital myotonic dystrophy in non-immune hydrops fetalis.

We report on the prenatal diagnosis of congenital myotonic dystrophy in a case of non-immune hydrops fetalis. DNA analysis of amniocytes revealed expansion of the trinucleotide (CTG) repeat within the myotonin PK gene, associated with myotonic dystrophy. The fetus was found to have approximately 1730 copies, while the normal population has between 5 and 27 copies, and minimally affected individuals have at least 50 copies.

Adult↗
PubMed1993

Wrist anatomy: incidence, distribution, and correlation of anatomic variations, tears, and arthrosis.

We dissected 393 wrists to evaluate the incidence and distribution of anatomic features, arthrosis, chondromalacia, and soft tissue lesions. The data were then analyzed for any statistically significant associations among the different variables. The most common (73%) lunate morphology had a separate medial facet on its distal surface for the hamate. The capitate had a separate facet for the fourth metacarpal in 86% of the wrists. Fourth metacarpals with a dorsal radial facet, either separate from or connected to the rest of the fourth metacarpal base, were the most common types of fourth metacarpal. Cartilage erosion with exposed subchondral bone was identified in 58% of the wrists. It was most commonly at the proximal pole of the hamate (28%). Tears of the ligaments and/or the triangular fibrocartilage complex were identified in 56% of the wrists. The triangular fibrocartilage complex was found torn in 36% of the wrists. The lunotriquetral interosseous ligament was torn in 36% of the wrists, and the scapholunate interosseous ligament was torn in 28% of the wrists. There was a communication between the proximal wrist joint and the pisotriquetral joint in 88% of the 76 wrists, which were further dissected to assess this issue. Statistical analysis of the data found a significant correlation between the presence of cartilage erosion at the proximal pole of the hamate and the presence of a lunate facet. There was also a significant correlation between the presence of a tear in the scapholunate interosseous ligament and the presence of cartilage erosion in the scaphoid-trapezium-trapezoid joint. Analysis of the paired wrists from 169 cadavers revealed that the same soft tissue tear or combination of tears was present bilaterally in 39% of the pairs. Cartilage erosion was present bilaterally in the same location or locations in 27% of the pairs.

Adolescent↗
PubMed1993

Load mechanics of the midcarpal joint.

The midcarpal joint was studied in upper extremities of fresh cadavers with the use of a static positioning frame, pressure-sensitive film, and a microcomputer to analyze the contact prints. The contact area on the proximal side of the midcarpal joint was found to consist generally of four areas; the scaphoid-trapezium-trapezoid, the scaphoid-capitate, the lunate-capitate, and the triquetrum-hamate. The contact areas accounted for less than 40% of the available joint surface, even under loads of 118 pounds. The distribution of load through the midcarpal joint was scaphoid-trapezium-trapezoid 23%, scaphoid-capitate 28%, lunate-capitate 29%, and triquetrum-hamate 20%. The midcarpal joint, like the radiocarpal joint, appears to transmit load through distinct areas and through a relatively small portion of the available joint surface.

Biomechanical Phenomena↗
PubMed1993

Measurement of carpal bone geometry by computer analysis of three-dimensional CT images.

The aim of this project was noninvasively to analyze and quantitate the geometry, load transfer characteristics, and spatial relationships of the carpal bones by using a new three-dimensional CT scan reconstruction technique. The determination of mechanical parameters such as distances between centroids and between bone surfaces, carpal alignment, volumes, surface areas, and contact areas can provide the basis for comparison between normal wrists and wrists with a variety of progressive instability patterns, types of fracture, pathologic and posttraumatic states, and different simulated surgical procedures. This new technology has demonstrated a volumetric accuracy of 94% and a linear accuracy of 97%. Simultaneous analysis of all articulating surfaces of multiple joints can be performed in cadavers and in patients because of the noninvasive nature of the imaging reconstruction technique. This new research offers much more information than has previously been available. It also promises direct application to the clinical setting and eliminates several limitations and questions that were inescapable with previous technology.

Adult↗