Biomedical subjects

M Locke

Publications and source records attributed to M Locke.

At least 37 records · Page 2Linked to original sources

PubMed1996

Increased HSF activation in muscles with a high constitutive Hsp70 expression.

Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5' region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of the rat hindlimb which differ in the constitutive expression of Hsp70, were assessed for HSF activation following heat shock. Mobility shift gel analyses demonstrated that HSF activation was detectable in extracts from all muscles following heat shock regardless of Hsp70 content. However, muscles comprised predominantly of slow/Type I fibers (soleus) demonstrated a greater HSF activation, as well as a faster HSF activation and inactivation, than muscles comprised predominantly of fast/Type II fibers (white gastrocnemius). In addition, muscles pretreated by two heat shocks (24 h apart) demonstrated a stronger HSF activation than muscles subjected to only one heat shock. Thus, results from cell culture models demonstrating that tissue levels of Hsp70 are inversely correlated with HSF activation, may not apply to the muscles of an intact animal.

Animals↗

PubMed1996

Diminished heat shock response in the aged myocardium.

Induction of heat shock proteins (Hsps), Hsp72 in particular, has been associated with myocardial protection. Since a decreased Hsp response has been reported to occur with aging, it was of interest to determine if hearts from aged animals also demonstrate an altered heat shock response and subsequent myocardial protection. Adult (6 months old) and aged (22 months old) Fischer 344 rats were heat stressed by raising their rectal temperatures to 41 degrees C for 10 min. At selected times following heat stress (0-24 h) hearts were examined for heat shock transcription factor trimerization and DNA-binding activity (Hsf1 activation), Hsp72 mRNA accumulation, Hsp72 and Hsf1 protein content, as well as, protection from ischemia using the Langendorff isolated heart model. Following heat stress, hearts from aged animals demonstrated a 47% reduction in Hsf1 activation, a reduction in Hsp72 mRNA and a 35% reduction in Hsp72 protein content, compared to hearts from adults. Interestingly, myocardial Hsf1 protein content was similar between aged and adult animals. Hearts from heat stressed adult animals (24-h prior) demonstrated an enhanced postischemic recovery as indicated by a greater recovery of left ventricular pressure and rate of contraction (P < 0.05), while hearts from heat stressed aged animals failed to demonstrate an enhanced postischemic recovery. These results suggest that hearts from aged animals exhibit an impaired ability to produce the protective Hsps and thus, may explain, at least in part, the increased susceptibility of aged hearts to stress.

Aging↗

PubMed1995

Heat shock protein (HSP 72) expression in patients undergoing cardiac operations.

The major mammalian stress-inducible protein, heat shock protein 72, protects cells from certain stresses and rapidly accumulates in cells after ischemia. Heat shock protein 72 is rapidly synthesized in the myocardium of various species in response to ischemia, but it has not been investigated in human heart. To determine if heat shock protein 72 accumulated in the ischemic myocardium of patients undergoing cardiac operations, we obtained sequential right atrial biopsy specimens from 12 patients undergoing repair at three intervals: before bypass, after reperfusion, and after bypass. Immunoblot analysis for heat shock protein 72 demonstrated a high expression in the human heart compared with other mammalian hearts, p (Binomial) = 0.01. Compared with before bypass, heat shock protein 72 contents after reperfusion and after bypass were 98.2% +/- 8.9%, p (signed-rank) = 0.65, and 87.6% +/- 17.1%, p (signed-rank) = 0.28, respectively. Although heat shock protein 72 concentration was unchanged in hearts after reperfusion and after bypass, the initial prebypass level of heat shock protein 72 was high. The high heat shock protein 72 level detected in human hearts may reflect preoperative disease and drug therapy, or inherently high levels may be usual in the human myocardium. These findings indicate that the myocardium of patients undergoing cardiac operations contains relatively high concentrations of heat shock protein 72, which are not increased during the surgical procedure.

Adolescent↗

PubMed1995

Transgenic canola and soybean seeds with increased lysine.

We have increased the lysine content in the seeds of canola and soybean plants by circumventing the normal feedback regulation of two enzymes of the biosynthetic pathway, aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes encoded by the Corynebacterium dapA gene and a mutant E. coli lysC gene, respectively, were linked to a chloroplast transit peptide and expressed from a seed-specific promoter in transgenic canola and soybean seeds. Expression of Corynebacterium DHDPS resulted in more than a 100-fold increase in the accumulation of free lysine in the seeds of canola; total seed lysine content approximately doubled. Expression of Corynebacterium DHDPS plus lysine-insensitive E. coli AK in soybean transformants similarly caused several hundred-fold increases in free lysine and increased total sed lysine content by as much as 5-fold. Accumulation of alpha-amino adipic acid (AA) in canola and saccharopine in soybean, which are intermediates in lysine catabolism, was also observed.

Aspartate Kinase↗

PubMed1995

Myocardial self-preservation: absence of heat shock factor activation and heat shock proteins 70 mRNA accumulation in the human heart during cardiac surgery.

Following myocardial ischemia, heat shock proteins (HSPs) have been found to be associated with a reduction in infarct size and enhanced postischemic functional recovery. Stress-induced regulation of the HSPs is mediated by the activation and binding of the heat shock transcription factor (HSF) to a specific DNA sequence located in front of all HSP genes, known as the heat shock element (HSE). To determine whether HSPs were induced in the human heart following the ischemic stress experienced during cardiac surgery, biopsies were performed of the right atrium at three sequential times: prior to establishing cardiopulmonary bypass; immediately after aortic declamping; and following termination of bypass. These samples from the atria of patients undergoing coronary bypass surgery were assessed for HSF activation using mobility shift gels, and analyzed for HSP 72 mRNA by Northern blot. Although a high level of the HSP 72 protein was noted at all intervals, no HSF activation was detected, nor was an accumulation of HSP 72 mRNA observed at any time during surgery. These data suggest that HSPs are not induced during cardiac surgery and that the high "constitutive" level of the HSP 72 protein detected in these hearts may not be secondary to an HSF-HSE interaction, but rather, the result of other transcription factors acting at alternative regions of the HSP 70 promoter.

Aged↗

PubMed1995

Cardiomyoplasty: preservation of the latissimus dorsi muscle.

Muscle necrosis has been frequently observed in cardiomyoplasty patients and in experimental animal studies. The purpose of this study was to determine if heat shock could provide protection to skeletal muscle as has been shown in cardiac muscle. A 15-minute heat shock at 42 degrees C resulted in an immediate increase in HSP72 mRNA and was followed within 3 hours by a two-fold increase in HSP72. Surgical dissection of the latissimus dorsi muscle (LDM) followed by an ischemic period resulted in a two-fold increase in HSP72 in control LDM, whereas the already high levels in the heat-shocked LDM increased only slightly with surgery and ischemia. Citrate synthase activity and tissue histology indicated that heat shock did not protect the LDM from the imposed surgical trauma and ischemia insults used in this study.

Animals↗

PubMed1995

Stress proteins: the exercise response.

A class of proteins that undergoes preferential synthesis following a variety of stressors has been demonstrated to carry out important cellular functions under both stressed and nonstressed conditions. These so-called heat shock (HSP) or stress (SP) proteins have been termed "molecular chaperones" and play important roles in cellular transportation, assembly/degradation, and cell survival. This review provides a basic introduction to the function and regulation of these proteins. Emphasis is placed on members of the HSP 70 family of proteins (especially HSP 72) and their role in cellular protection, their pattern of distribution in skeletal muscle, and changes in their expression following exercise and exercise training.

Animals↗

PubMed1995

Activation of heat-shock transcription factor in rat heart after heat shock and exercise.

Stress-induced transcriptional regulation of the heat-shock proteins (HSP) is mediated by activation and binding of the heat-shock transcription factors (HSF) to the heat-shock element (HSE). Given the similarities between the stressors known to activate the HSF in cultured cells and the physiological stresses known to occur during exercise, HSF activation was examined in the hearts from exercising animals. Sprague-Dawley rats (5 rats/group) were run on a treadmill (24 m/min) for either 0, 20, 40, or 60 min or to exhaustion (102 +/- 7 min). Protein extracts were assessed for HSF activation by mobility-shift gels. Extracts from the hearts of nonrunning rats demonstrated no HSF activation, whereas HSF activation was detected in 80% of the hearts from animals that run for at least 40 min. These results demonstrate that treadmill running is capable of activating the HSF and increasing 70-kDa HSP mRNA in the rat myocardium.

Animals↗

PubMed1995

Enhanced postischemic myocardial recovery following exercise induction of HSP 72.

The inducible isoform of the 70-kDa heat shock protein (HSP) family, HSP 72, has been shown to protect cells from protein-damaging stressors and has been associated with myocardial protection. Because exercise is capable of increasing HSP 72 content, we determined whether exercise induction of HSP 72 also provided myocardial protection. Twenty-eight rats (n = 7 per group) were divided into control, heat-shocked (15 min at 42 degrees C), and two exercised groups. Exercise consisted of either one or three bouts (on 3 consecutive days) of treadmill running for 60 min at 30 m/min. Twenty-four hours after heat shock or exercise, hearts were placed on a Langendorff apparatus and subjected to 30 min of global ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP), maximal rate of contraction and relaxation (+/- dP/dt, respectively), coronary flow, catalase activity, and HSP 72 content were determined. During reperfusion, hearts from heat-shocked animals and animals subjected to three bouts of exercise recovered a greater percentage of preischemic LVDP and +/- dP/dt compared with controls or animals that exercised only once. Compared with hearts from controls, HSP 72 content was significantly elevated in the hearts of heat-shocked animals and in animals subjected to three bouts of exercise, but not in animals that exercised only once. These results suggest that exercise induction of HSP 72 can confer an enhanced postischemic recovery and may explain, at least in part, the myocardial protection associated with exercise.

Animals↗

PubMed1994

Preservation and contrast without osmication or section staining.

Conventional treatment of tissues for sectioning and transmission electron microscopy uses aldehyde fixation and osmium tetroxide postfixation. Although the result is aesthetically pleasing, osmication destroys some cell components and reduces the chemical activity of others, such as reaction with antibodies and lectins. We have found that aldehyde fixation followed by uranyl acetate preserves and contrasts most structures and visualizes some that are not easily seen after osmication. Aldehyde/UA treated tissues have enough contrast to be observed without section staining while retaining some of the chemical activity that is lost through osmication. Sections of tissues with good preservation and contrast can be used for immunogold and lectin-gold labelling of at least some molecules.

Aldehydes↗

PubMed1994

The cuticular localization of integument peptides from particular routing categories.

The distribution of integument peptides in relation to chitin and structural features has been studied in the surface epidermis of the caterpillar of Calpodes ethlius by immunoblotting and immunogold labelling using antibodies prepared to peptides isolated from lamellate endocuticle or from hemolymph. The intermoult cuticle consists of an epicuticle, an endocuticle of many chitin containing lamellae, and a chitin containing assembly zone directly above the apical epidermal microvilli and the perimicrovillar space. During the intermoult, the epidermis secretes peptides constitutively, that is, secretory vesicles containing peptides exocytose without accumulating, traverse the perimicrovillar space and form lamellae in the assembly zone. At moulting, the epidermis deposits ecdysial droplets in addition. These interrupt the last few lamellae which later go on to become the perforated ecdysial membrane. The integument is involved with four routing classes of peptide. Secretion is apical into the cuticle (C), basal into the hemolymph (H), bidirectional (BD), or transported to the cuticle across the epidermis from the hemolymph (T). Some peptides change their routing at moulting. There are several patterns of localization. (1) C and BD cuticular peptides occur mainly in chitin containing lamellate cuticle. (2) Some are also present in epicuticle, and are therefore not obligatorily linked to chitin or matrix between chitin fibers. Cuticular peptides that also occur in the hemolymph are glycosylated, whereas most that are only secreted apically into the cuticle are not. All BD but few C peptides carry alpha-D-glucose/alpha-D-mannose. Some C and BD peptides carry N-acetyl glucosamine. (3) C36 extracted from cuticle has most N-acetyl glucosamine and colocalizes with chitin rather than the protein matrix. It is therefore probably the main link between chitin fibers and the matrix. (4) H235 is barely detectable at the apical cell surface during the intermoult but is abundant at moulting around and below the ecdysial droplets. (5) T66 occurs in intermoult lamellate cuticle. At moulting, alone among the peptides examined, it is in ecdysial droplets. Intermoult C and BD peptides are not in ecdysial droplets but continue to be present in the ecdysial membrane, suggesting that constitutive secretion is independent from the exocytosis of transported moult peptides. T66 differs from most hemolymph peptides in that it does not carry N-acetyl glucosamine or alpha-D-glucose/alpha-D-mannose. (6) Weakly reacting BD peptides (and some H peptides barely detectable in cuticle) localize near the apical surface. Their distribution therefore favours apical secretion and retrieval as a mechanism for basal secretion.

Animal Structures↗

PubMed1994

Shifts in type I fiber proportion in rat hindlimb muscle are accompanied by changes in HSP72 content.

Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.

Animals↗

PubMed1994

Resection arthrodesis of the knee for tumor: large intercalary allograft and long intramedullary nail technique.

Twenty-five patients had a procedure consisting of resection of the distal femur or proximal tibia for aggressive or malignant neoplasms. The resulting bony gap was bridged by allografts averaging 18 cm in length. Fixation was achieved using a long fluted intramedullary nail. The average follow-up time and time to union were 3 years (range, 0.25 to 9 years) and 13 months (range, 6 to 28 months), respectively. Eleven patients had nononcologic complications related to the index surgical procedure, including two patients with allograft nonunion. There were no infections. Three patients had amputations for local recurrence of their tumor. Twenty patients (80%) had no evidence of tumor at the time of follow-up. According to the Evaluation System of the Musculoskeletal Tumor Society, 80% of patients had a satisfactory result. No patient had a nononcologic complication resulting in total failure or amputation. The procedure of resection-arthrodesis of the knee using a large allograft with a long intramedullary nail provides patients with a stable, durable biological reconstruction that allows early weight-bearing, and the procedure carries a low incidence of infection.

Adolescent↗

PubMed1993

The planes of division in twin cell doublets.

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells remain connected by residual midbodies between mitoses. These twins resemble one another more than their other neighbours in features such as the shape and number of nucleolar particles, the number of actin bundles, the position of condensed chromosomes in female cells and the timing of mitosis. Although the patterns of arrangement of structures may be similar in twinned nuclei they differ in their orientation. We have now found that twins also differ in the orientation of their division planes with respect to the previous plane of division. Similarity of structural pattern but not orientation is most easily explained if nuclei, together with determinants for the plane of division, are free to rotate in the plane of the epithelium.

Animals↗

PubMed1993

The timing of division in twin cell doublets.

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells are twins connected by residual midbodies between divisions. Twin cells resemble one another more than their other neighbours in such features as the shape and number of nucleolar particles, the number of actin bundles and the position of condensed female chromosomes. We have now found that they also resemble one another in the timing of cell division in preparation for pupation. Twins are more likely to divide together than at random. The paired timing of mitosis is presumed to be a result of the twins sharing a common cytoplasm.

Animals↗

PubMed1991

Inducible isoform of HSP70 is constitutively expressed in a muscle fiber type specific pattern.

The most prominent group of stress or heat-shock proteins (HSPs) has an Mr of approximately 70,000 and is collectively referred to as the HSP70 family. The extent of stress inducibility and subcellular location of the various HSP70 isoforms differ, but all appear to be involved with ATP-dependent stabilization or solubilization of proteins. One isoform, termed the inducible isoform of HSP70 (HSP72i), is normally absent in unstressed cells. In a previous study, we detected a protein corresponding in Mr and pI to HSP72i in unstressed rat muscle. Therefore, it was of interest to determine if this expression in unstressed muscle cells is general or confined to specific muscle fiber types. To answer this question we have employed various rat hindlimb muscles that differ in fiber type proportion from predominantly type I (soleus) to predominantly type IIB (white gastrocnemius). Proteins from muscle homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to a nylon membrane, probed with a monoclonal antibody for HSP72i, and visualized using an alkaline phosphatase-conjugated secondary antibody. Immunoblot analyses demonstrate the constitutive expression of HSP72i in rat muscles comprised primarily of type I muscle fibers (soleus), but not in muscles comprised primarily of type IIB fibers (white gastrocnemius). In muscles of mixed fiber type, HSP72i content is roughly proportional to the percentage of type I fibers. These results substantiate that unstressed rat muscles express the inducible HSP72 isoform and demonstrate that its constitutive expression is proportional to the type I muscle fiber composition.

Animals↗

PubMed1991

The visualization of apoferritin in the secretory pathway of vertebrate liver cells.

Electron microscopy has shown that normal mouse liver cells contain abundant cytosolic and lysosomal holoferritin but none in cisternae of the rough endoplasmic reticulum (RER). This does not mean that ferritin is absent from the secretory pathway, since the low contrast of apoferritin and ferritin containing little iron makes it difficult to resolve using standard transmission electron microscopy. We hypothesized that treatment of permeabilized cells and cell fractions with iron should make apoferritin visible by converting it to holoferritin. The iron treatment caused particles resembling the cores of holoferritin to appear in RER and in RER microsomes from mouse liver. We conclude that ferritin occurs in the endoplasmic reticulum and could be secreted, allowing the liver to be a source of serum ferritin.

Animals↗

PubMed1991

Paired cytoskeletal patterns in an epithelium of siamese twin cells.

The epidermis of some insects is a sheet of siamese twin cells which are formed by conserving the midbody between siblings after cell division. We have found that for about 36 h after ecdysis to the 5th stage, the cells of Calpodes caterpillars contain one to five or more actin bundles. The variation in number of bundles occurs in an epithelium that is presumed to be otherwise genetically and developmentally homogeneous. The number of bundles is paired in adjacent cells (P less than 0.005, n = 617). Confocal microscopy shows midbodies between paired but not between unpaired cells. The pairing is reminiscent of the paired nucleolar patterns in these siamese twin cells (Locke, M., H. Leung, Tissue and Cell 17, 573-588 (1985)) or the mirrored patterns of stress fibers in newly divided 3T3 cells (Albrecht-Buehler, G., J. Cell Biol. 72, 595-603 (1977)). The pairing provides further evidence for the operation of transiently heritable factors as determinants for cell pattern.

Actins↗