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Biomedical subjects

R W Hendrix

Publications and source records attributed to R W Hendrix.

At least 19 recordsLinked to original sources
PubMed1995

Complexes between chaperonin GroEL and the capsid protein of bacteriophage HK97.

The 42 kDa capsid protein of bacteriophage HK97 requires the GroEL and GroES chaperonin proteins of its Escherichia coli host to facilitate correct folding, both in vivo and in vitro. In the absence of GroES and ATP, denatured gp5 forms a stable complex with the 14 subunit GroEL molecule. We characterized the electrophoretic and biochemical properties of this complex. In electrophoresis on a native (nondenaturing) gel, the band of the gp5-GroEL complex shifts to a slower migrating position relative to uncomplexed GroEL. The results show that there is only one subunit of gp5 bound to each GroEL 14-mer and that the shift in band position is due primarily to a change in the overall charge of the complex relative to uncomplexed GroEL, and not to a change in size or shape. GroEL forms similar complexes with proteolytic fragments of gp5, with a series of sequence duplication derivatives of gp5, and with other proteins. Electrophoretic examination of these complexes shows that a band shift occurs with proteins larger than 31-33 kDa but not with smaller proteins. For those proteins that cause a band shift upon complex formation, the magnitude of the shift is correlated with the predicted if the charge of the complex were simply the sum of the charge of GroEL and the charge of the substrate protein. We suggest that binding of a substrate protein to GroEL is accompanied by a net binding of solution cations to the complex, but only in the case of proteins above a minimum size of 31-33 kDa. The gp5-GroEL complex is in an association/dissociation equilibrium, with a binding constant measured in the range of 11-17 microM-1.

Adenosine Triphosphate↗
PubMed1995

Assembly in vitro of bacteriophage HK97 proheads.

Bacteriophage HK97 is a lambdoid phage with a head assembled from 415 copies of a 42 kDa subunit arranged in an icosahedrally symmetrical lattice with a triangulation number of 7. Prohead I, the first shell structure in the assembly pathway, is composed of 42 kDa coat protein subunits that have not yet undergone the proteolytic cleavage, conformational changes, and covalent cross-linking steps that occur later in the assembly of mature heads. Prohead I can be efficiently dissociated into capsomeres by treatment with 2 M KCl. The resulting capsomeres are a mixture of two species, identified as pentamers and hexamers of the 42 kDa subunit. These capsomeres were also detected as the products of chaperonin-assisted renaturation of 42 kDa polypeptide in vitro at room temperature or in the course of self folding and assembly in vitro at 0 degrees C. Pentamer and hexamer capsomeres can be interconverted in vitro by manipulating solvent conditions, and this makes it possible to carry out the in vitro shell assembly reaction at different input ratios of hexamer to pentamer. The Prohead I structures produced are always the normal (T = 7) size regardless of the input pentamer to hexamer ratio. Assembly is most efficient when the pentamer to hexamer ratio is 1:5 (a mass ratio of 1:6), or the same as the capsomere ratio in a T = 7 shell.

Capsid↗
PubMed1995

Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system.

Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").

Amino Acid Sequence↗
PubMed1995

Genetic basis of bacteriophage HK97 prohead assembly.

We report studies to determine which bacteriophage genes are required for assembly of phage HK97 proheads and what roles they play. We identify the gene encoding the major capsid protein of phage HK97 and report its DNA sequence, together with the DNA sequences of the two genes immediately upstream from it. When the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of the unprocessed 42 kDa capsid protein. No separately encoded scaffolding protein is required for this assembly. If the 25 kDa product of the next gene upstream is co-expressed with the capsid protein, the prohead structures that are produced undergo the normal morphogenetic cleavage, which removes 102 amino acids from the N terminus of each subunit, leaving 31 kDa subunits. The 25 kDa protein is therefore probably a phage-encoded protease. The third gene, upstream from the protease gene, encodes the portal protein. Presence of the portal protein is not required for assembly of the capsid protein. Analysis of the phenotypes of four single amino acid-substitution mutants in the capsid-protein gene leads to several insights into the functions of the capsid protein and its interactions with the putative protease.

Amino Acid Sequence↗
PubMed1995

Structural transitions during bacteriophage HK97 head assembly.

Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

Amino Acid Sequence↗
PubMed1995

Bacteriophage HK97 head assembly.

The head assembly pathway of bacteriophage HK97 shares many features with head assembly pathways determined for other dsDNA phages, and it also provides examples of novel variations on the basic theme. We describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. Comparisons of head assembly pathways among different phages, as well as comparisons of the organization of the genes that specify those pathways, suggest the range of different solutions phages have found to common assembly problems and give insight into the evolutionary histories of these assembly processes.

Bacteriophages↗
PubMed1992

Bacteriophage lambda PaPa: not the mother of all lambda phages.

The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

Adsorption↗
PubMed1992

Computed tomography of posterior fracture-dislocations of the shoulder: case reports.

Computed tomographic (CT) evaluation of seven posterior shoulder dislocations in five patients is reported. Computed tomography provided better visualization of the trough fracture in the humeral head than did conventional x-ray films or tomography. It also demonstrated fracture fragments not seen on conventional roentgenograms. The two cases of bilateral dislocation and one case of unilateral dislocation were caused by seizures. The two other cases of unilateral dislocation were caused by trauma.

Adult↗
PubMed1992

Anterior cruciate ligament injury: MR imaging diagnosis and patterns of injury.

The anterior cruciate ligament (ACL) is an important stabilizer of knee motion. Injury of the ACL can lead to substantial disability; an accurate diagnosis of ACL injury is vital in both short-term and long-term patient care. Magnetic resonance (MR) imaging has emerged as the study of choice to evaluate the status of the ACL and other associated structures in the knee. Sagittal MR images have been commonly used in the evaluation of the ACL. However, the authors believe that coronal and axial imaging planes can add useful information about ACL injury and, thus, lead to improved accuracy and confidence regarding diagnosis. Multiplanar imaging can readily demonstrate meniscal, ligamentous, and bone marrow injuries that commonly occur with the most frequent mechanisms of ACL injury. These mechanisms, in order of frequency, include internal rotation and valgus stress, hyperextension, and varus stress with external rotation. An understanding of these mechanisms is helpful in the MR diagnosis of ACL injury.

Anterior Cruciate Ligament Injuries↗
PubMed1991

Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits.

We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation.

Amino Acid Sequence↗
PubMed1991

Cortical bone metastases.

The bone scans and radiographs of 27 patients with solitary or multiple cortical bone metastases were retrospectively studied. Thirty-six cortical metastases were identified in 32 appendicular long bones. Origin of the tumors included lung, breast, kidney, pancreas, larynx, uterus, and site unknown. Cortical metastases have hitherto been reported as individual cases or in small numbers. The large number reported herein suggests that the occurrence of these lesions may be more common than previously expected. A cortical metastasis was the only evidence of metastatic disease in eight of the 27 patients.

Aged↗
PubMed1990

Case report 620. Progressive systemic sclerosis (PSS) with paraspinous and intraspinous calcifications.

Progressive systemic sclerosis (PSS) is a connective tissue disorder of unknown etiology and pathogenesis characterized by fibrosis of the skin (scleroderma) and variable involvement of several organ systems including the gastrointestinal tract, lungs, heart, and kidneys. Calcinosis cutis is a frequent finding, commonly seen in the soft tissues of the fingertips and over the knees and elbows. Calcifications may also occur in areas of muscle necrosis and have been reported in articular synovium. This report describes a patient with PSS in the form of subcutaneous calcifications and scleroderma who was noted to have extensive paraspinous and intraspinal calcific deposits, causing severe narrowing of the spinal canal. The radiographs of 66 patients with PSS seen at Northwestern Memorial Hospital of soft tissue calcifications.

Calcinosis↗
PubMed1990

Evaluating the multiply injured patient radiographically.

In the multiply injured patient there are obvious lesions that often overshadow other lesions, creating a significant possibility that they will be overlooked. Because of the high incidence of missed lesions in such patients, it is a good idea to approach the patient with the presumption that one may be overlooking something and ask oneself what other lesions may be associated with the known lesions. We have enumerated several injuries that frequently occur together and have called them clinical dyads. It is hoped that knowledge of the associations will prevent oversight of the second lesion. The radiographic examination serves as an extension of the physical examination, confirming clinical suspicions and documenting the extent of many injuries. We have outlined what we believe is a prudent radiographic examination in a multiply injured patient. It provides information about the areas most likely to have injuries but is not so extensive that it hinders patient care. It is a starting place or survey and may lead to other, more complicated radiographic studies should the findings warrant them.

Communication↗
PubMed1989

Diagnostic imaging of fracture complications.

Fracture complications as discussed in this article are those abnormalities occurring as a direct result of or in association with a fracture or dislocation. Complications with radiographic findings are stressed, although those without definite radiographic manifestations are mentioned for the sake of completeness.

Angiography↗
PubMed1989

A study of glenohumeral orientation in patients with anterior recurrent shoulder dislocations using computerized axial tomography.

Thirty-six patients with recurrent anterior shoulder dislocations and 11 normal patients who had no history of shoulder problems were evaluated by a G.E. 8800 fourth generation computerized axial tomographer to quantitate humeral head retrotorsion and glenoid tilt. The study objective was to determine if osseous orientation was a major etiologic factor in patients with recurrent anterior shoulder dislocations. The normal position of the humeral head as determined by CT scan is 30 degrees retroversion with respect to its distal articular axis. In dislocations this value changes to a more anteverted position of 24 degrees retroversion. Glenoid tilt could not be given a single value since it changes from retroversion superiorly toward more anteversion inferiorly. This change in glenoid tilt was greater in the dislocators with inferior sections measuring 2 degrees anteversion in the dislocators and 1 degree retroversion in the uninvolved shoulder and in the normal population. The change in humeral retroversion and glenoid tilt in the recurrent dislocators was statistically significant when compared with the uninvolved shoulder and to the normal population. Thus, the glenoid humeral orientation appears to be a significant predisposing factor to recurrent anterior shoulder dislocations.

Adult↗
PubMed1988

Homologous plant and bacterial proteins chaperone oligomeric protein assembly.

An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.

Adenosine Triphosphatases↗