Changes In Isoforms Research Articles

Assessment of ovarian reserve by using the follicle-stimulating hormone isoform distribution pattern to predict the outcome of in vitro fertilization

Changes in FSH isoforms occur at temporally specific times during the menstrual cycle and reproductive lifespan. Changes in ratio in FSH isoforms may provide a sensitive test of ovarian reserve before IVF.

Interleukin-1β Signals through a c-Jun N-Terminal Kinase-Dependent Inducible Nitric Oxide Synthase and Nitric Oxide Production Pathway in Sertoli Epithelial Cells

Our recent Sertoli cell (SC) studies showed that the c-Jun N-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of IL (IL-1alpha, IL-1beta, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1beta regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1beta mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SCs were maintained in serum-free cultures and treated with FSH (100 ng-1 microg/ml) or IL-1beta (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 microM) or JNK (SP600125, 10 microM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species were measured by flow cytometry and ELISA. IL-1beta transiently induces intracellular NO (30 min) but not reactive oxygen species. Subsequently, iNOS mRNA and protein expression (3-6 h) significantly increased after IL-1beta but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive endothelial NOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1beta-induced iNOS expression and NO production. Such findings suggest that intra- and extracellular NO within the tubule may alert SCs monitoring the microenvironment to an aberrant cytokine, triggering antioxidant and antiinflammatory activities to avoid disruption of spermatogenesis.

Effects of exercise and creatine on myosin heavy chain isoform composition in patients with Charcot–Marie–Tooth disease

It is not known whether myosin heavy chain (MHC) content changes in response to exercise training or creatine supplementation in subjects with Charcot-Marie-Tooth disease (CMT). Based on previous data, we hypothesized that resistance exercise and creatine would increase the percentage of type I MHC composition in the vastus lateralis muscle and that myosin isoform changes would correlate with improved chair rise-time in CMT subjects. To test this hypothesis, 18 CMT subjects were randomly assigned to either a placebo or creatine group. All subjects performed a 12-week, home-based, moderate-intensity resistance training program. Chair rise-time was measured before and after the training program. Muscle biopsies were obtained from the vastus lateralis before and after the 12-week program. Gel electrophoresis showed a significant decrease (approximately 30%) in MHC type I in CMT subjects given creatine supplementation when compared with placebo. There was a nonsignificant increase in both MHC type IIa (approximately 23%) and MHC type IIx (approximately 7%) in CMT subjects given creatine. Reduced MHC type I content and increased MHC type IIa content correlated with faster chair rise-times (i.e., improved muscle performance). The training-induced change in MHC IIa content was inversely correlated with chair rise-time in CMT subjects given creatine. When the two subject groups were combined, there was a linear, negative relationship between the change in MHC type IIa content and chair rise-time after training and a positive relationship between the training-induced change in MHC type I content and chair rise-time. These data suggest that improved function (chair rise-time) was associated with a lower level of MHC type I and increased MHC type IIa composition. Furthermore, the data are consistent with the hypothesis that creatine supplementation alters MHC composition in CMT patients undergoing resistance training and that MHC changes associated with creatine supplementation can improve muscle function.

CYP1A-immunopositive proteins in bivalves identified as cytoskeletal and major vault proteins

To identify possible CYP1A-immunopositive proteins in bivalves, we used anti-fish CYP1A antibodies combined with one- and two-dimensional gel electrophoresis and mass spectrometry, and found that two of the main CYP1A-immunopositive proteins in digestive gland of Mytilus edulis, were cytoskeletal actin (42 kDa) and major vault protein (102 kDa), while the main CYP1A-immunopositive protein in the clam Chamaelea gallina was the cytoskeletal protein tropomyosin (33 kDa). Anti-CYP1A cross-reactive bands of 48–54 and 75 kDa in M. edulis were observed but not identified in this study. Sequence alignments with one of the most conserved CYP1A regions (NIRDITDSLIDHCED) from fish revealed similarities with tropomyosin and actin sequences from mussels, which could explain the immunological cross-reactivity. Changes in isoforms of tropomyosin after exposure to Aroclor1254 and Cu(II), could indicate modifications due to oxidative stress. Effects of pollutant related oxidative stress on the cytoskeleton require further studies.

Functional consequence of mutation in rat cardiac troponin T is affected differently by myosin heavy chain isoforms

Cardiac troponin T (cTnT) is an essential component of the thin filament regulatory unit (RU) that regulates Ca2+ activation of tension in the heart muscle. Because there is coupling between the RU and myosin crossbridges, the functional outcome of cardiomyopathy-related mutations in cTnT may be modified by the type of myosin heavy chain (MHC) isoform. Ca2+ activation of tension and ATPase activity were measured in muscle fibres from normal rat hearts containing alpha-MHC isoform and propylthiouracil (PTU)-treated rat hearts containing beta-MHC isoform. Muscle fibres from normal and PTU-treated rat hearts were reconstituted with two different mutations in rat cTnT; the deletion of Glu162 (cTnT(E162DEL)) and the deletion of Lys211 (cTnT(K211DEL)). Alpha-MHC and beta-MHC isoforms had contrasting impact on tension-dependent ATP consumption (tension cost) in cTnT(E162DEL) and cTnT(K211DEL) reconstituted muscle fibres. Significant increases in tension cost in alpha-MHC-containing muscle fibres corresponded to 17% (P < 0.01) and 23% (P < 0.001) when reconstituted with cTnT(E162DEL) and cTnT(K211DEL), respectively. In contrast, tension cost decreased when these two cTnT mutants were reconstituted in muscle fibres containing beta-MHC; by approximately 24% (P < 0.05) when reconstituted with cTnT(E162DEL) and by approximately 17% (P = 0.09) when reconstituted with cTnT(K211DEL). Such differences in tension cost were substantiated by the mechano-dynamic analysis of cTnT mutant reconstituted muscle fibres from normal and PTU-treated rat hearts. Our observation demonstrates that qualitative changes in MHC isoform alters the nature of cardiac myofilament dysfunction induced by mutations in cTnT.

Maintenance of slow type I myosin protein and mRNA expression in overwintering prairie dogs (Cynomys leucurus and ludovicianus) and black bears (Ursus americanus)

Hibernating mammals have the remarkable ability to withstand long periods of fasting and reduced activity with dramatic maintenance of skeletal muscle function and protein composition. We investigated several hindlimb muscles of white-tailed prairie dogs (Cynomys leucurus) and black bears (Ursus americanus), two very different hibernators who are dormant and fasting during winter. The black-tailed prairie dog (C. ludovicianus) remains active during winter, but suffers minor skeletal muscle atrophy; nevertheless, they also demonstrate apparent skeletal muscle adaptations. Using SDS-PAGE, we measured myosin protein isoform profiles before and after the hibernation season. All species maintained or increased levels of slow myosin, despite the collective physiological challenges of hypophagia and reduced activity. This contrasts markedly with standard mammalian models of skeletal muscle inactivity and atrophy predicting significant loss of slow myosin. A mechanism for changes in myosin isoforms was investigated using reverse-transcription PCR, following partial sequencing of the adult MHC isoforms in C. leucurus and U. americanus. However, mRNA expression was not well correlated with changes in MHC protein isoforms, and other synthesis and degradation pathways may be involved besides transcriptional control. The muscles of hibernating mammals demonstrate surprising and varied physiological responses to inactivity and atrophy with respect to slow MHC expression.

Changes in lipoxygenase isoforms during germination and early seedling growth of Pisum sativum L.

Investigations of physiological functions of seed lipoxygenase during germination and early seedling growth are complicated by the presence of multiple isoforms with different product specificity. The combination of isoelectric focusing, lipoxygenase in-gel activity staining and mass spectrometry showed that only two seed lipoxygenases, LOX-2 and LOX-3, were active at neutral pH in protein extracts from dry and germinating pea seeds (Pisum sativum L.), with LOX-3 more abundant than LOX-2. Each active seed LOX appeared at multiple pIs, probably resulting from post-translational modifications. The abundance of pea seed lipoxygenases was lower in embryonic shoots than in other parts of the axis. After radicle emergence, seed LOXs were degraded, as revealed by Western blotting using antibodies specific to pea seed LOXs. Two new vegetative isoforms, LOX-N3 and LOX-g, appeared in the shoot and gradually replaced the pea seed lipoxygenases as the principal contributors to LOX enzyme activity during early seedling growth. Activities of LOX-N3 and LOX-g were abundant in shoot tips, but not root tips, in pea seedlings. Thus, during germination, there is a shift of LOX activity from radicles to shoots that accompanies the transition from seed LOXs to vegetative LOXs. These results confirmed that identification of pea LOXs based solely on pI may be misleading; however, mass spectrometry enabled more accurate identification of these highly homologous proteins to be made.

Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica

Ramonda sp. (Gesneriaceae) is an endemic and relic plant in a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. Senescence- and drought-related changes in the activity of peroxidase (POD; EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), and superoxide dismutase (SOD; EC 1.15.1.1) were determined in leaves of different age and relative water content. The results indicate that different POD isoforms were stimulated during senescence and dehydration. Two of the soluble POD isoforms were anionic with pI 4.5, and two were cationic with pI 9.3 and 9.0. The activity of ascorbate peroxidase remained unchanged either by drought or senescence. For the first time, SOD isoforms have now been determined in this resurrection plant. Several SOD isoforms, all of the Mn type, were found to be anionic with pI 4 and a few others had pI from 5 to 6, while one band of FeSOD with a lower molecular weight was neutral. Rehydration brought about a remarkable decrease over the first hour in the activity of all the antioxidant enzymes examined but activity recovered 1 d after rehydration. The results confirmed that dehydration and senescence caused disturbance in the redox homeostasis of Ramonda leaves, while inducing different POD isoforms. A physiological role of peroxidase reaction with hydroxycinnamic acids in conservation and protection of cellular constituents of desiccated Ramonda leaves is suggested.

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.

Temporal Response of Desmin and Dystrophin to Progressive Resistance Exercise

PURPOSE: Our purpose was to investigate the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 weeks of progressive resistance training and following a single bout of unaccustomed resistance exercise. METHODS: Muscle biopsies were taken from the vastus lateralis of 12 untrained men a week prior to exercise (week 0) and at weeks 1,2,4, and 8 following a single bout of exercise (n = 6), or at weeks 1, 2, 4 and 8 of the progressive resistance training program (n = 6). Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS PAGE at each time point for each group. RESULTS: In the training group, the desmin:actin ratio significantly increased compared to week 0 beginning at week 4 (0.054 to 0.095, P < 0.0001) and continuing through week 8 (0.054 to 0.094, P < 0.0001). The desmin:actin ratio did not change at any time point for the single bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5719+/−382 and 6582+/−640, weeks 0 and 4 respectively, P < 0.05). Finally, a significant negative correlation was observed between the desmin:actin ratio and the percentage of MHC IIx (R = −0.31, P < 0.05, all time points from both groups). CONCLUSION: These data suggest a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but prior to changes in MHC isoforms, while dystrophin remains unchanged.

Change in the Actin Cytoskeleton during Seismonastic Movement of Mimosa pudica

The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement.

Conformational changes in Nitric Oxide Synthase Isoforms Under High Hydrostatic Pressure

We studied the effects of high hydrostatic pressure on the overall protein structures and the bound flavins of three isoforms of the wild type nitric oxide synthase. Under a pressure gradient of 1 bar to 2.5 kbar, nNOS and eNOS show a decrease in size (compression) when monitored by light scattering, whereas iNOS shows an increase. In the holo proteins and hemedomains of nNOS and eNOS, the effect is reversible when pressure is released. In the presence of H4B, both nNOS and eNOS show significant decreases from their initial sizes after depressurization. For all the isoforms, the kinetics of the flavin fluorescence intensities at constant pressure of 2 kbar, show an intial increase, after which they decrease in a biphasic manner. The compression rates, as followed by light scattering were faster than the flavin distortion, as followed by fluorescence. Assay of NO synthesis by the NOS isoforms, as measured by hemoglobin capture, showed, that the electron transfer abilities of NOS were mostly retained when it was subjected to 2.5 kbar and depressurized soon after the rising phase of flavin intensities reached a maximum (~5–7 min). The decreasing phases showed very slow kinetics, taking ~2–3 hr for completion, depending upon the isoform. The depressurized NOS isoforms at the completion of slow kinetic phase were completely inactive. Based on the results from the pressure induced changes in the flavin conformations, a model for the changes in the electron transfer abilities of the NOS isoforms is proposed. (Supported by grants NIH GM52419 and HL30050 to BSM).

Proteomics of the Heart

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide new insights into cellular mechanisms involved in cardiac dysfunction. The majority of proteomic investigations still use 2D gel electrophoresis (2-DE) with immobilized pH gradients to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. It can resolve >5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect <1 ng of protein per spot. Furthermore, 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. The use of proteomics to investigate heart disease should result in the generation of new diagnostic and therapeutic markers. In this article, we review the current status of proteomic technologies, describing the 2-DE proteomics workflow, with an overview of protein identification by MS and how these technologies are being applied to studies of human heart disease.

Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

Transcriptional disturbance is implicated in the pathology of polyglutamine diseases, including Huntington's disease (HD). However, it is unknown whether transcriptional repression leads to neuronal death or what forms that death might take. We found transcriptional repression-induced atypical death (TRIAD) of neurons to be distinct from apoptosis, necrosis, or autophagy. The progression of TRIAD was extremely slow in comparison with other types of cell death. Gene expression profiling revealed the reduction of full-length yes-associated protein (YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD. Furthermore, novel neuron-specific YAP isoforms (YAPΔCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPΔCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPΔCs also markedly attenuated Htt-induced neuronal death in primary neuron and Drosophila melanogaster models. Collectively, transcriptional repression induces a novel prototype of neuronal death associated with the changes of YAP isoforms and p73, which might be relevant to the HD pathology.

Changes in host chitinase isoforms in relation to wounding and colonization by Heterobasidion annosum: early and strong defense response in 33-year-old resistant Norway spruce clone

We studied the defense reactions of 33-year-old susceptible and resistant clones of Norway spruce (Picea abies (L.) Karst.) to the major root-rot fungus Heterobasidion annosum (Fr.) Bref. and determined if tissue cultures can be used as a model system for studying defense responses of mature trees at the molecular level. Quantitative PCR analysis of genomic DNA obtained from samples taken at different times along the lesion length in living bark indicated that the fungus was present in higher amounts and extended further into the host tissue in the susceptible clone than in the resistant clone. In protein extracts from the same lesion samples, there were differences in temporal and spatial changes in host chitinase isoform profiles between the resistant and susceptible clones. Host chitinase isoforms with pI values approximately 4.8, 4.4 and 3.7 increased more during the first 7 days after wounding and inoculation and extended further along the lesion length in the resistant clone than in the susceptible clone. These results suggest that the time from wounding and infection to induction of defense-related expression is shorter in the resistant clone indicating a more efficient host defense response than in the susceptible clone. Tissue cultures from the same clones were not resistant to H. annosum and showed no difference in the timing of the increase in chitinase isoforms in response to the pathogen. However, tissue cultures from both clones showed an increase in chitinase isoforms within 6 to 24 h past inoculation, indicating that increased chitinase expression in response to the pathogen is part of a general defense response common to both mature clones and tissue cultures.

Hypothyroid Phenotype Is Contributed by Mitochondrial Complex I Inactivation Due to Translocated Neuronal Nitric-oxide Synthase

Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-alpha (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3',5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype.

Temporal response of desmin and dystrophin proteins to progressive resistance exercise in human skeletal muscle

We have investigated the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 wk of progressive resistance training or after a single bout of unaccustomed resistance exercise. Muscle biopsies were taken from the vastus lateralis of 12 untrained men. For the single-bout group (n=6) biopsies were taken 1 wk before the single bout of exercise (week 0) and 1, 2, 4, and 8 wk after this single bout of exercise. For the training group (n=6), biopsies were taken 1 wk before the beginning of the program (week 0) and at weeks 1, 2, 4, and 8 of the progressive resistance training program. Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS-PAGE at each time point for each group. In the training group, desmin was significantly increased compared with week 0 beginning at week 4 (182% of week 0; P<0.0001) and remained elevated through week 8 (172% of week 0; P<0.0001). Desmin did not change at any time point for the single-bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single-bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and it further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5,719+/-382 and 6,582+/-640 microm2, weeks 0 and 4, respectively; P<0.05). Finally, a significant negative correlation was observed between the desmin-to-actin ratio and the percentage of MHC IIx (R=-0.31; P<0.05, all time points from both groups). These data demonstrate a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but before changes in MHC isoforms, whereas dystrophin remains unchanged.

The effects of short-term sprint training on MCT expression in moderately endurance-trained runners

The purpose of this study was to assess the effects of short-term sprint training on transient changes in monocarboxylate lactate transporter 1 (MCT1) and MCT4 protein and mRNA content. Seven moderately endurance-trained runners (mean +/- SE; age 27.7+/-2.9 years, body mass 81.1+/-5.9 kg, .VO(2max) 58.1+/-2.0 ml kg(-1) min(-1)) completed a .VO(2max) and a supramaximal running test to exhaustion (RTE) before and after a 6-week period of sprint training. The sprint training was progressive and consisted of 18 sessions of near maximal short duration (5-15 s) sprints to compliment the athlete's endurance training. Prior to the training period there was a significant (P<0.05) increase in MCT1, but not MCT4 protein, 2 h after the RTE. This occurred without any change in corresponding mRNA levels. After the training period, there was a significant increase in MCT1 protein but no significant change in the MCT4 isoform. Both MCT1 and MCT4 mRNA was significantly lower at rest and 2 h post-RTE after the completion of the training period. After the training period, there was a significant increase in the time to exhaustion and distance covered during the RTE. This study demonstrates that sprint training of this length and type results in an upregulation of MCT1 protein, but not MCT4 content. Additionally, this study shows conflicting adaptations in MCT1 and MCT4 protein and mRNA levels following training, which may indicate post-transcriptional regulation of MCT expression in human muscle.

Ovine serum and pituitary isoforms of luteinising hormone during the luteal phase

The relative abundance of the different isoforms of pituitary and circulating luteinising hormone (LH) in ewes, at different times after the administration of gonadotrophin-releasing hormone (GnRH), during the luteal phase of the oestrous cycle was investigated. Sixteen ewes on Day 9 of their cycle were divided into four groups (n = 4). The control group (T0) received saline solution; the remaining animals received 100 microg GnRH (i.m.) 30, 90 or 180 min (T30, T90 and T180, respectively) before serum and pituitary gland collection. Luteinising hormone polymorphism was analysed by chromatofocusing (pH 10.5-3.5). The LH eluted from each chromatofocusing was grouped on the basis of the following three criteria: (1) according to the pH of elution (pH > or = 10-3.5); (2) as either a basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic (pH < or = 6.4) elution of LH of serum and hypophyseal origin; and (3) on the basis of distinct isoforms, of which 10 (A-J) were identifiable in hypophyseal extracts and four (A-D) were found in the serum. In general, the most abundant forms of LH in both the pituitary and serum, at all times, were basic. However, that proportion was greater in hypophyseal extracts (84 +/- 3%, 81 +/- 4%, 82 +/- 3% and 83 +/- 2% at T0, T30, T90 and T180, respectively) than in serum (51 +/- 5%, 48 +/- 10% and 54 +/- 6% at T30, T90 and T180, respectively). Neutral and acidic LH made up a larger proportion of the total LH in sera (neutral: 17 +/- 4%, 20 +/- 6% and 23 +/- 3% at T30, T90 and T180, respectively; acidic: 32 +/- 8%, 32 +/- 11% and 23 +/- 6% at T30, T90 and T180, respectively) than in the pituitary extracts (neutral: 4.0 +/- 0.7%, 10 +/- 4%, 7 +/- 2% and 5.0 +/- 0.5% at T0, T30, T90 and T180, respectively; acidic: 12 +/- 3%, 11 +/- 2%, 12 +/- 2% and 12 +/- 2% at T0, T30, T90 and T180, respectively) at all times. These data reveal that the relative composition of the LH present in the pituitary gland and the LH secreted into the circulation is different, with more neutral and acidic isoforms being secreted. The pattern of circulating LH isoforms changes between 30 and 180 min after GnRH peak induction, with a greater proportion of isoform C (eluting between pH 7.0 and 6.5) at T180 compared with T30 and T90.

Changes in laminin isoforms associated with brain tumor invasion and angiogenesis

Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, alpha, beta and gamma. More than 15 laminin isoforms are known to date and the expression of specific isoforms may change in certain pathological conditions. Here we show that during progression of glial tumors laminin-9 (alpha4beta2gamma1) is switched to laminin-8 (alpha4beta1gamma1), which is dramatically increased in glial brain tumors. Laminin-8 overproduction by glial tumor cells facilitates spread of glioma. Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time. Laminin-8 may be thus used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.