Proportion Of Isoforms Research Articles

Power Output and Muscle Myosin Heavy Chain Composition in Young and Elderly Men

Aging is associated with a decline in muscle volume, power output, and the velocity at which peak power (Vopt) occurs. The current study aimed to examine the relationship between lower-limb power output characteristics, muscle myosin heavy chain (MHC) composition, and lean limb volume. Lower-limb power output during repeated efforts on an inertial sprint cycle and single-leg thrusts on the modified Nottingham power rig was studied in seven young and seven old males in relation to muscle MHC isoform composition of the vastus lateralis. Older subjects produced significantly lower power outputs and Vopt under all conditions (P < 0.01) and had lower proportions of fast MHC isoforms (P< 0.05). Peak power output during cycling was significantly related to lower-limb lean volume (r = 0.92, P < 0.05), whereas Vopt during sprint cycling was closely related to vastus lateralis MHC-II composition (r = 0.80, P < 0.05). These results provide further evidence of the importance of fast myosin isoform composition in the maintenance of dynamic muscle function in later life and particularly for maximal cycling performance.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant neurodegenerative disorder, which has three cardinal features: behavioral and personality changes, cognitive impairment, and motor symptoms. FTDP-17 was defined during the International Consensus Conference in Ann Arbor, Michigan, in 1996. The prevalence and incidence remain unknown but FTDP-17 is an extremely rare condition. It is caused by mutations in the tau gene, which encodes a microtubule-binding protein. Over 100 families with 38 different mutations in the tau gene have been identified worldwide. The phenotype of FTDP-17 varies not only between families carrying different mutations but also between and within families carrying the same mutations. The pathogenetic mechanisms underlying the disorder are thought to be related to the altered proportion of tau isoforms or to the ability of tau to bind microtubules and to promote microtubule assembly. Definitive diagnosis of FTDP-17 requires a combination of characteristic clinical and pathological features and molecular genetic analysis. Genetic counseling should be offered to affected and at-risk individuals; for most subtypes, penetrance is incomplete. Currently, treatment for FTDP-17 is only symptomatic and supportive. The prognosis and rate of the disease's progression vary considerably among individual patients and genetic kindreds, ranging from life expectancies of several months to several years, and, in exceptional cases, as long as two decades.

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater ( p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F ( p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% ( p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% ( p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.

Different apoprotein(a) isoform proportions in serum and carotid plaque

Introduction Cardio- and/or cerebro-vascular risk are associated with high lipoprotein (a) [Lp(a)] levels and low-molecular-weight (LMW) apo(a) isoforms. Aims of this study were to evaluate the deposition of apo(a) isoforms and apoprotein B (apo B) in atherosclerotic plaque from patients (males and females) who had carotid endarterectomy for severe stenosis, and to identify differences between patients classified by gender and divided according to the stability or instability of their plaques. Materials and methods We determined lipids, apo B and Lp(a) in serum and plaque extracts from 55 males and 25 females. Apo(a) was phenotyped and isoforms were classified by number of kringle IV (KIV) repeats. Results Lp(a) levels were higher in female serum and plaque extracts than in male samples, while apo B levels were lower. More Lp(a) than apo B deposition was observed in plaque after normalization for serum levels. Thirty-one different apo(a) isoforms were detected in our patients, with a double band phenotype in 94% of cases. In both sexes, the low/high (L/H) molecular weight apo(a) isoform expression ratio was significantly higher in plaque than in serum. Females with unstable plaques had higher Lp(a) levels in both serum and tissue extracts, and fewer KIV repeats of the principal apo(a) isoform in the serum than the other female group or males. Conclusions In both sexes, the same apo(a) isoforms are found in serum and atherosclerotic plaque, but in different proportions: in plaque, LMW apo(a) is almost always more strongly accumulated than HMW apo(a), irrespective of any combination of apo(a) isoforms in double band phenotypes or Lp(a) serum levels. Moreover, serum and tissue Lp(a) levels were higher in females than in males, and particularly in the group with unstable plaques.

Parasternal intercostal muscle remodeling in severe chronic obstructive pulmonary disease

Studies in experimental animals indicate that chronic increases in neural drive to limb muscles elicit a fast-to-slow transformation of fiber-type proportions and myofibrillar proteins. Since neural drive to the parasternal intercostal muscles (parasternals) is chronically increased in patients with severe chronic obstructive pulmonary diseases (COPDs), we carried out the present study to test the hypothesis that the parasternals of COPD patients exhibit an increase in the proportions of both slow fibers and slow myosin heavy chains (MHCs). Accordingly, we obtained full thickness parasternal muscle biopsies from the third interspace of seven COPD patients (mean +/- SE age: 59 +/- 4 yr) and seven age-matched controls (AMCs). Fiber typing was done by immunohistochemistry, and MHC proportions were determined by SDS-PAGE followed by densitometry. COPD patients exhibited higher proportions of slow fibers than AMCs (73 +/- 4 vs. 51 +/- 3%; P < 0.01). Additionally, COPD patients exhibited higher proportions of slow MHC than AMCs (56 +/- 4 vs. 46 +/- 4%, P < 0.04). We conclude that the parasternal muscles of patients with severe COPD exhibit a fast-to-slow transformation in both fiber-type and MHC proportions. Previous workers have demonstrated that remodeling of the external intercostals, another rib cage inspiratory muscle, elicited by severe COPD is characterized by a slow-to-fast transformation in both fiber types and MHC isoform proportions. The physiological significance of this difference in remodeling between these two inspiratory rib cage muscles remains to be elucidated.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

The Human Muscle Proteome in Aging

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.

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.

Classification of human liver transplant recipients by their preoperative CD8+ T cell subpopulation and its relation to outcome

The primed status of T cells is markedly different among liver transplant recipients, due to a lifetime of antigen exposure and reduced thymopoiesis by aging, and diseases. This study aims to characterize the preoperative immunological status of CD8+ T cell subpopulations and relate it to the outcome for liver transplant recipients. We classified 112 liver transplant recipients into 5 groups, based on hierarchical clustering of the CD8+CD45 isoform proportion of T cells. In Groups I and II (pediatric), the naive T cell proportion was more than 50%. In adult recipients, Group III was characterized by a naive T cell proportion of 50%, Group IV had the greatest effector/memory T cells (EM), and Group V had the greatest proportion of effector T cells. In Groups IV and V, the effector T cell proportion was considerably higher, and was accompanied by marked downregulation of the CD27+CD28+ subsets and upregulation of interferon gamma (IFN)-gamma, tumor necrosis factor-alpha, and perforin expression. Group V recipients tended to be complicated postoperatively, with a significantly reduced survival rate (1 yr, 66.8%) and markedly reduced Eastern Cooperative Oncology Group performance status.

Pituitary–ovarian axis during lactational amenorrhoea. II. Longitudinal assessment of serum FSH polymorphism before and after recovery of menstrual cycles

The association of normal serum levels of immunoassayable gonadotrophins with anovulation during lactational amenorrhoea (LA) has not been fully explained. Serum FSH polymorphism was analysed in 10 women during LA between days 60 and 70 post-partum and again, in the mid-follicular phase (MFP), after resuming menstrual cyclicity. FSH microheterogeneity was characterized according to charge, using preparative isoelectric focusing, and according to the inner structure of carbohydrate chains, using lectin chromatography. A significantly higher proportion of FSH charge isoforms isolated below pH 4.10 and a lower proportion of FSH isoforms bearing highly branched oligosaccharides were observed during LA when compared to MFP. Further analysis with higher resolution showed that FSH charge isoforms, isolated in the lower pH range in LA, corresponded to FSH molecules bearing highly branched and biantennary oligosaccharides. FSH isoforms bearing hybrid-type oligosaccharides were only present during LA. The circulating FSH isoform mix was significantly less bioactive in LA than in MFP. LA is characterized by a more acidic mix of FSH isoforms, containing hormone bearing less processed oligosaccharides, with decreased biopotency in comparison with the follicular phase. This FSH microheterogeneity may be one of the critical factors contributing to incomplete follicular development and anovulation during LA.

Effect of Deficits in Laryngeal Sensation on Laryngeal Muscle Biochemistry

Swallowing deficits in elderly people are significant clinical problems and may be associated with impaired pharyngolaryngeal sensation. However, the extent to which sensory innervation affects the motor system is unclear. Our purpose was to examine differences in biochemical properties of laryngeal muscles following sensory nerve ablation. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate laryngeal muscles of young and old Fischer 344/Brown Norway rats, and rats that underwent sensory ablation via bilateral section of the superior laryngeal nerve, internal branch (SLNi), or mixed sensory-motor nerve ablation via left-sided recurrent laryngeal nerve (RLN) section. In lateral thyroarytenoid muscle, a reduction was found in the proportion of the most rapidly contracting myosin heavy chain isoform (type 2B) with SLNi section, RLN section, and aging. Section of the SLNi did not alter the proportion of any myosin heavy chain isoform within the lateral cricoarytenoid or posterior cricoarytenoid muscles, but RLN section resulted in a reduction in the proportion of type 2B. Accordingly, alteration in biochemical properties of the lateral thyroarytenoid muscle alone was demonstrated following sensory ablation. We conclude that sensory changes may affect properties of laryngeal muscles, and may thus have an impact on motor control during critical functions, such as airway protection during swallowing.

Alterations in the Proportions of Skeletal Muscle Proteins following a Unilateral Lesion to the Substantia Nigra Pars Compacta of Rats

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30-70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of alpha-tropomyosin and increased the proportion of beta-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson's disease may be associated with alterations to the expression of skeletal muscle proteins.

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

We investigated the expression and functional properties of slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight (sTnT1, sTnT2) and low-molecular-weight (sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca(2+) activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca(2+) affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.

Circulating non-22 kDa growth hormone isoforms after a repeated GHRH stimulus in normal subjects

The aim of this study was to evaluate the proportion of non-22 kDa GH isoforms in relation to total GH concentration after a repeated GHRH stimulus in healthy subjects. We studied 25 normal volunteers (12 males and 13 females, mean age 13.1 years, range 6–35), who received two GHRH bolus (1.5 μg/kg body weight, i.v.) administered separately by an interval of 120 minutes. The proportion of non-22 kDa GH was determined by the 22 kDa GH exclusion assay (GHEA), which is based on immunomagnetic extraction of monomeric and dimeric 22 kDa GH from serum, and quantitation of non-22 kDa GH isoforms using a polyclonal GH assay. Samples were collected at baseline and at 15–30 min intervals up to 240 min for total GH concentration. Non-22 kDa GH isoforms were measured in samples where peak GH after GHRH was observed. Total GH peaked after the first GHRH bolus in all subjects (median 37.2 ng/ml; range: 10.4–94.6). According to GH response to the second GHRH stimulus, the study group was divided in “non-responders” ( n = 7; 28%), with GH peak levels lower than 10 ng/ml (median GH: 8.7 ng/ml; range 7.3–9.6) and “responders” ( n = 18; 72%), who showed a GH response greater than 10 ng/ml (median 17 ng/ml; range 10.1–47.0). The median proportion of non-22 kDa GH on the peak of GH secretion after the first GHRH administration was similar in both groups (“responders” median: 8.6%, range 7–10.9%; “non-responders” median: 8.7%, range 6.7–10.3%), independently of the type of response after the second GHRH. In contrast, the median proportion of non-22 kDa GH was greater at time of GH peak after the second GHRH bolus in the “non-responders” (median 11.4%; range 9.1–14.3%) in comparison with the “responders” (median 9.1%; range 6.7–11.9%; p = 0.003). A significant negative correlation between the total GH secreted and the percentage of non-22 kDa isoforms was seen in the “non-responders” ( p = 0.003). These differences in GH response to repeated GHRH stimulation and in the pattern of GH isoforms at GH peak among subjects might be due to distinct recovery patterns of somatrotrophic function and/or differences in metabolic clearance of GH isoforms.

Proteomics analysis of growth hormone isoforms in the human pituitary

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0°C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.

Impact of plasmid supercoiling on the efficacy of a rabies DNA vaccine to protect cats

As of today, most DNA vaccination trials have been performed with plasmid preparations highly enriched in supercoiled molecules (sc) and the importance of supercoiled versus open circular (oc) plasmid isoforms for vaccine immunogenicity has only received limited attention. This study demonstrated that a single rabies DNA vaccination fully protected cats against a lethal rabies challenge as early as 3 weeks post vaccination provided that the proportion of supercoiled isoform in the vaccinal solution is at least 48%. In contrast, vaccination with a plasmid containing only 20% of supercoiled molecules induced significant but only partial protection. Further, a single rabies DNA vaccination with plasmids containing at least 70% of supercoiled molecules triggered statistically significant specific antibody titers and specific Th-1 oriented cell-based immunity as early as 2 and 3 weeks post vaccination, respectively. It is concluded that the oc isoforms are less efficient than supercoiled isoforms at inducing a complete profile of immune responses. Therefore, it is proposed that the target threshold of supercoiling that must be met by a rabies DNA vaccine to guarantee optimal immune responses and protection, be set at 70% of supercoiled molecules in the vaccine solution.

Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers

The pattern of distribution of circulating luteinizing hormone (LH) isoforms in cattle during estrus and the luteal phase was investigated. In each stage, the stage of the estrous cycle was synchronized in seven Holstein heifers with a prostaglandin analogue. After estrus was detected, blood samples were taken at 2-h intervals for 24 h. In the luteal phase, animals received 250 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. LH concentration in the samples was determined. Samples with the greatest LH concentration in estrus (pre-ovulatory peak) and those collected 60 min after GnRH administration (luteal phase) were analyzed by chromatofocusing, eluted with a pH gradient from 10.5 to 3.5. Eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both phases, basic forms were the most abundant, and these were greater ( P < 0.05) during the luteal phase (78.4 ± 4.2%) as compared with during estrus (57.1 ± 6.2%); the proportion of neutral and acidic isoforms in estrus (13.7 ± 2.6%; 28.5 ± 2.8%) was greater ( P < 0.05) as compared with the luteal phase (3.0 ± 0.7; 18.7 ± 3.4). These results indicate that the relative proportion of LH isoforms secreted by the adenohypophysis differ by stage of estrous cycle. The addition of excess of NaCl to the column modifies the antigen–antibody binding in the RIA, and the proteins eluted are erroneously quantified as LH; this is an artifact of the technique.

Effects of Short-Term Concentric vs. Eccentric Resistance Training on Single Muscle Fiber MHC Distribution in Humans

The purpose of this investigation was to determine the effects of a concentric vs. eccentric resistance training program on single muscle fiber myosin heavy chain (MHC) adaptations in humans. Fifteen sedentary, healthy males were divided into three groups: concentric training (CTG) (n = 6, 24.2 +/- 1.7 y, 181 +/- 2 cm, 82.5 +/- 4.6 kg), eccentric training (ETG) (n = 6, 23.7 +/- 1.6 y, 178 +/- 3 cm, 90.4 +/- 6.1 kg), and control (CTL) (n = 3, 23 +/- 1.5 y, 181 +/- 2 cm, 97 +/- 13.2 kg). The subjects performed 4 sets of 8 unilateral repetitions starting at 80 % of concentric 1-RM, 3 days/week for a total of 4 weeks. Subjects were tested pre- and post-training for concentric 1-RM. Muscle biopsies were obtained from the vastus lateralis pre- and post-training for determination of single fiber MHC isoform distribution using SDS-PAGE/silver staining (100 fibers analyzed/subject pre- and post-training). Fibers expressing more than one MHC isoform (i.e., hybrid fibers) were analyzed for relative MHC isoform proportions via densitometry. The training program resulted in a 19 % 1-RM strength gain for CTG (p < 0.05) with no change in ETG or CTL. MHC-IIx fibers decreased by 7 % in CTG (p < 0.05) and ETG had an 11 % increase in total hybrids (MHC-I/IIa + MHC-IIa/IIx) (p < 0.05). No other differences were noted in MHC distribution among the three groups. Densitometry analysis of hybrid fibers showed no change in relative MHC isoform proportions pre- to post-training for any group. These data suggest that the MHC distribution did not change dramatically as a result of 4 weeks of concentric vs. eccentric resistance training despite the increase in whole muscle strength from concentric muscle actions.

Sulfatide and Na+-K+-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na(+)-K(+)-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na(+)-K(+)-ATPase activity, especially in Na(+)-K(+)-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na(+)-K(+)-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na(+)-K(+)-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na(+)-K(+)-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na(+)-K(+)-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.