Effect of nutrient intake on intramuscular glucose metabolism during the early growth stage in cross-bred steers (Japanese Black male × Holstein female).

The objective was to investigate the impact of nutrient intake during the early growth period on the expression of glucose metabolism-related genes in skeletal muscle of cross-bred cattle. From 1.5 to 5 months of age, group H (n=7) animals were intensively fed a high-protein and low-fat milk replacer [crude protein (CP) 28%; ether extracts (EE) 18%; max: 2.0 kg, 12 l/day], and group R (n=7) animals were fed a restricted amount of normal milk replacer (CP 25%; EE 23%; max 0.5 kg, 4 l/day). From 6 to 10 months of age, group H cattle were fed a high-nutrition total mixed ration mainly prepared from grain feed, and group R cattle were fed only roughage. Blood samples were taken from each animal at three biopsy times (1.5, 5 and 10 months of age), and the blood plasma concentration of glucose and insulin was analysed. In glucose concentration, there were no significant differences; however, the concentrations of insulin were higher in group H than in group R at 5 and 10 months of age. Muscle samples were taken by biopsy from longissimus thoracis muscle (LT) at 1.5, 5 and 10 months of age. We analysed mRNA expression levels using the quantitative real-time polymerase chain reaction (PCR) assay for glucose transporters (GLUT1 and GLUT4), insulin receptor, phosphatidylinositol 3-kinase (PI-3K), protein kinase B (PKB, also known as Akt), hexokinase 1 (HK1) and tumour necrosis factor alpha (TNFα). Although no differences were detected at 1.5 and 5 months of age, at 10 months of age, GLUT1, HK1 and TNFα mRNA expression levels were significantly higher in group H than in group R. These results suggested Glut1 that affects insulin-independently mediated glucose uptake was more responsive to improved nutrition during early growth stage than GLUT4 that insulin-dependently mediated glucose uptake in LT of cattle.

Technical note: Determination of cell-specific gene expression in bovine skeletal muscle tissue using laser microdissection and reverse-transcription quantitative polymerase chain reaction.

Skeletal muscle is a very heterogeneous tissue consisting of diverse cell types with specific transcription profiles. Therefore, the measured mRNA abundance of a certain cell type marker is influenced by the transcriptional activity as well as by the usually unknown number of contributing cells in the sample. In studies on the transcriptional activity of adipogenic genes, as indicators for the development of intramuscular adipocytes, an altered number of adipocytes or respective progenitor cells can mask changes in transcriptional activity. To overcome this problem, we started to use laser microdissection to isolate RNA of adipocytes and muscle fibers separately for downstream analysis. Even muscle fiber types can be collected and analyzed separately. Laser microdissection in combination with biopsy techniques enables gene expression studies of particular cell types during the life cycle of an animal. First experiences using laser microdissection for adipogenic gene expression studies in bovine skeletal muscle are described, and the influence of sample preparation and future challenges are discussed.

Cellular conditions for intramuscular fat deposition in Japanese Black and Holstein steers.

The experiment was conducted to study the development of intramuscular fat in Japanese Black (JB) compared to Holstein (HS) steers and to find breed differences for fat depot development and distribution in the carcass under equal feeding conditions. Additional to slaughter samples, biopsy samples of longissimus muscle (LM) and subcutaneous fat, taken at 10, 14, 18, and 22 months of age, were used for histological and molecular investigations. Japanese Black steers stored about 14% more fat in the LM (P = 0.001), resulting in larger marbling flecks (P < 0.001). Muscle fibers and intramuscular adipocytes in both breeds responded to the high energy feeding with significant enlargement, which was faster in JB. Histograms of intramuscular adipocytes size showed a shift toward larger cells during growth, but also the abundance of small, developing adipocytes. This development was accompanied by a correlated up-regulation of adipogenic genes until 22 months of age.

Synergistic cytotoxic effects in myeloid leukemia cells upon cotreatment with farnesyltransferase and geranylgeranyl transferase-I inhibitors.

As deregulation of RAS signaling is important in the pathogenesis of myeloid leukemias, molecular targeting of RAS signaling may be a promising therapeutic strategy. Farnesyl transferase inhibitors (FTIs) are the most promising class of these new cancer therapeutics. Several FTIs have entered phase II clinical trials in acute myeloid leukemia (AML). Since geranylgeranylation of K-RAS and N-RAS in the presence of FTIs may represent an important mechanism of FTI resistance, 6 geranylgeranyl transferase-I inhibitors (GGTIs) were screened alone and in combination with FTI for growth inhibition of myeloid leukemia cells. Significant growth inhibition (>70%) in myeloid cell lines was observed for GGTI-286 (9/19), GGTI-298 (14/19), GGTI-2147 (16/19) and FTI L-744,832 (17/17). GGTI treatment of NB-4 cells resulted in an accumulation of cells in G(0)/G(1), whereas FTI L-744,832 primarily caused an increase in G(2)/M. FTI and GGTIs both induced apoptosis. In all cases, FTI/GGTI cotreatment led to synergistic cytotoxic effects in both myeloid cell lines (5/5) and primary AML cells (6/6). This synergy coincided with increased apoptosis. FTI/GGTI cotreatment caused an accumulation of unprocessed N-RAS and inactive N-RAS-RAF complexes. Our results suggest that alternative geranylgeranylation of N-RAS may represent an important mechanism of resistance to FTI monotherapy in myeloid leukemia cells.

Feeding daidzein to late pregnant sows influences the estrogen receptor beta and type 1 insulin-like growth factor receptor mRNA expression in newborn piglets.

The present study was undertaken to determine the tissue-specific expression of estrogen receptor beta (ERbeta), and the effects of a daidzein supplement to the diet of pregnant sows on the expression of ERbeta, and type 1 insulin-like growth factor receptor (IGF-1R) genes in newborn piglets by using semi-quantitative RT-PCR. Eight sows received a dietary supplement of daidzein at a dosage of 8 mg per kg feed from day 85 of gestation, and six sows were used as controls. After parturition, 2 male neonatal piglets were selected from each litter for sampling. ERbeta mRNA was detected in intestine, lung, thymus, kidney, pituitary and hypothalamus tissues, but not in heart, adrenal, skeletal muscle, liver or placental tissues. Daidzein treatment significantly increased the birth weight of male piglets and markedly reduced the level of ERbeta mRNA in the hypothalamus, but not in the pituitary. An up-regulation of IGF-1R gene transcription was observed in skeletal muscles of newborn piglets. In addition, the IGF-1R mRNA was found to be most abundant in pituitary and hypothalamus, followed by skeletal muscle, thymus, and liver tissues in decreasing order. Our results demonstrate that (1) ERbeta is expressed in a tissue-specific manner in newborn piglets, (2) daidzein down-regulates ERbeta gene expression in the hypothalamus, possibly indicating central effects of daidzein, and (3) daidzein influences fetal growth associated with higher IGF-IR gene expression in skeletal muscle.

Isoflavones, substances with multi-biological and clinical properties.

Isoflavones, rich in soybean, are currently receiving much attention because of their potential role in preventing and treating cancer and other human chronic diseases. The present review provides an overview of the recent results in this research field. Data from epidemiological reports and laboratories have shown that isoflavones have multi-biological and pharmacological effects in animals and humans. These include estrogenic and antiestrogenic effects, cell signalling conduction, as well as cell growth and death. Based on these properties, soy protein and isoflavones have been associated with reduced incidences of breast and prostate cancers, cardiovascular diseases or osteoporosis, and exhibit some other favorable effects. The mechanism through which isoflavones may exert the above-mentioned functions are not only based on the estrogenic properties of isoflavones, but also on their role as protein tyrosine kinase inhibitors, as regulators of gene transcription, modulators of transcription factors, antioxidants, as well as by altering some enzyme activities. However, to draw a clear conclusion regarding the clinical use of isoflavones further investigation would be required, although only a few effects of short- or long-term use of soy proteins are known in humans.

Fat storage capacity in growth-selected and control mouse lines is associated with line-specific gene expression and plasma hormone levels.

OBJECTIVE: For a detailed understanding of the complex traits growth and fat storage, a dissection into single genetic entities is mandatory. Therefore, blood plasma concentrations of hormones and the expression of selected genes were measured in extremely differentiated mouse lines. Genes were selected as candidates which might influence the complex traits body weight and fat accumulation, and which are located in chromosomal regions recently identified to affect trait differences between the lines. SUBJECTS AND MEASUREMENTS: The mouse lines were selected for high body weight (DU6), high carcass protein content (DU6P) and unselected controls (DUKs). In the selected lines DU6 and DU6P, mean body weights at the age of six weeks were about twice as high as the DUKs, whereas total fat weight was increased 2.2-fold in DU6 mice compared to DU6P and 3.2-fold in comparison to DUKs. Blood plasma concentrations of insulin-like growth factor 1 (IGF-1), growth hormone (GH), insulin and leptin, were measured in all lines at three weeks and at six weeks of age. Expression patterns of the genes encoding growth hormone (Gh), insulin-like growth factor 1 (Igf1), lipoprotein lipase (Lpl), glycerolphosphate dehydrogenase 1 (GDC-1), and adipocyte protein 2 (Ap2) were analyzed by Northern blot hybridization. RESULTS: In line DU6, highly significant increased concentrations of insulin and leptin were observed at six weeks of age; at this stage, IGF-1 concentrations were elevated in the two selected lines compared to controls with maximal concentrations of IGF-1 and GH in DU6P. The amount of mRNA for GH in the pituitary gland, for Igf1 in the liver and for LPL in epididymal fat tissue was significantly elevated in the two selected lines compared to controls at the age of three weeks, but not at six weeks. IGF-1 and GDC-1 mRNA concentrations were significantly higher in the DU6 mice than in the DU6P (P < 0.01) and the DUKs (P < 0.001) mice examined at both ages. CONCLUSIONS: The results prove line-specific concentrations of the analyzed hormones and the transcription amounts of Gh, Igf1, GDC-1 and Lpl. The measured differences are either direct genetic effects or secondary changes, resulting from different food consumption.

Post mortem changes in Ca2+ transporting proteins of sarcoplasmic reticulum in dependence on malignant hyperthermia status in pigs.

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined. The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.

Can spinal surgery be prevented by aggressive strengthening exercises? A prospective study of cervical and lumbar patients.

OBJECTIVE: To determine if patients recommended for spinal surgery can avoid the surgery through an aggressive strengthening program. SETTING: A privately owned clinic, staffed by physicians and physical therapists, that provides treatment for patients with neck and/or back pain. METHODS: Over a period of 2 1/2 years, consecutive patients referred to the clinic for evaluation and treatment were enrolled in the study if they (1) had a physician's recommendation for lumbar or cervical surgery, (2) had no medical condition preventing exercise, and (3) were willing to participate in the approximately 10-week outpatient program. Treatment consisted mainly of intensive, progressive resistance exercise of the isolated lumbar or cervical spine. Exercise was continued to failure, and patients were encouraged to work through their pain. Third-party payors in Minneapolis were surveyed for average costs. Average follow-up occurred 16 months after discharge. RESULTS: Forty-six of the 60 participants completed the program; 38 were available for follow-up and three required surgery after completing the program. DISCUSSION/CONCLUSIONS: Despite methodologic limitations, the results are intriguing. A large number of patients who had been told they needed surgery were able to avoid surgery in the short term by aggressive strengthening exercise. This study suggests the need to define precisely what constitutes "adequate conservative care."

Internucleosomal DNA fragmentation in ovine luteal tissue associated with luteolysis: in vivo and in vitro analyses.

Internucleosomal DNA fragmentation, a characteristic of apoptosis, can be visualized with agarose gel electrophoresis as discrete low-molecular-weight DNA fragments (laddering), in multiples of approximately 185 bp. CL were collected from superovulated ewes (control) or at 12 h after injection of prostaglandin F2 alpha (PGF2 alpha) on various days after hCG injection. The ability of PGF2 alpha on Days 8, 10, 12, and 14 (n > or = 3 per day per treatment) to induce luteal cell DNA fragmentation was evaluated. DNA was isolated and visualized on agarose gels. No DNA fragmentation was observed in CL from control ewes on Days 8, 10, or 12. Internucleosomal fragmentation of DNA (indicative of apoptosis) as well as nonspecific DNA fragmentation (indicative of non-apoptotic cell death) in CL from Day 14 controls was observed in two of four animals. Additionally, this pattern of DNA fragmentation was observed in CL from ewes treated with PGF2 alpha on all days. Evidence of DNA fragmentation was observed in luteal tissue after dissociation, yet no fragmentation was observed in unsliced, non-dissociated CL collected from Day 10 control ewes (incubated 4 h), or in sliced, non-incubated CL. Slicing and incubation alone were sufficient to initiate DNA fragmentation. A variety of approaches were utilized to inhibit DNA fragmentation. Only the addition of zinc acetate (1 mM) in the incubation medium throughout the 4-h incubation period prevented DNA fragmentation that was initiated by slicing (p < 0.05). There appear therefore, to be one or more intraluteal factors that directly initiate DNA fragmentation associated with cell death in luteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of Ca2+ homeostasis in ovine large and small luteal cells.

This study was undertaken to characterize differences in Ca2+ homeostasis between small and large ovine luteal cells. Although increasing extracellular pH (pHex) resulted in increases in intracellular calcium ([Ca2+]in) in both cell types, the large cells exhibited a greater sensitivity, suggesting that distinct [Ca2+]in regulatory mechanisms with distinct pH optima are operating in the two cell types. The sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA) increased [Ca2+]in in both cell types. Treatment of small cells with CPA resulted in transient increases in [Ca2+]in, whereas CPA produced sustained increases in [Ca2+]in in large cells. In small cells, pretreatment with CPA prevented further increases in [Ca2+]in in response to TG and vice versa. In large cells, TG pretreatment precluded further increases in [Ca2+]in with either prostaglandin F2 alpha (PGF2 alpha) or CPA. In contrast, after CPA pretreatment, PGF2 alpha or TG induced further increases in [Ca2+]in in large cells. This suggests that a TG-sensitive, CPA-insensitive Ca2+ pool is present in large cells but not in small cells. Neither Na+ removal nor KCl addition affected [Ca2+]in in either cell type, indicating that neither the Na+/Ca2+ exchanger nor voltage-dependent Ca2+ channels are involved in Ca2+ homeostasis in these cells. Addition of the calcium antagonist, LaCl3 (La3+), produced a gradual increase in [Ca2+]in in large cells but no changes in [Ca2+]in in small cells. Additionally, treatment with increasing concentrations of 4-bromo-A23187 resulted in titratable increases in [Ca2+]in that are greater in large than small cells, suggesting that small cells possess a higher Ca(2+)-buffering capacity than large cells. Progesterone secretion by large cells was significantly inhibited at alkaline pHex. In the presence of PGF2 alpha, progesterone secretion exhibited a distinct pH optimum of 7.0. In contrast, pHex did not affect secretion of progesterone in small cells under any of the conditions tested. TG, CPA, and La3+ all reduced secretion of progesterone in large, but not small, cells. These results demonstrate that ovine large and small luteal cells differ in regulation of [Ca2+]in homeostasis, and that treatments that increase [Ca2+]in decrease progesterone secretion in large cells but have no effect in small cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin F2 alpha releases calcium from a thapsigargin-sensitive pool in ovine large luteal cells.

Thapsigargin (TG) and A23187 were used to examine the regulation of cytosolic free calcium (Cai2+) in ovine large and small luteal cells. Thapsigargin (50 nM) induced a sustained increase of Cai2+ in fura 2-acetoxymethyl ester (AM)-loaded cells (large = 1.32 +/- 0.07-fold, small = 1.45 +/- 0.07-fold, P < 0.05). A23187 (1 microM) induced a rapid transient increase of Cai2+ (large = 1.37 +/- 0.07-fold, small = 1.46 +/- 0.10-fold, P < 0.05). In large cells, 0.5 microM prostaglandin F2 alpha (PGF2 alpha) increased Cai2+ 1.54 +/- 0.11-fold. Pretreatment with 50 nM TG abolished the PGF2 alpha-induced calcium response. Pretreatment with PGF2 alpha attenuated (P < 0.05) the TG-induced Cai2+ increase. Progesterone secretion was significantly (P < 0.05) inhibited by incubation with 50 nM TG, 1 microM A23187, and 0.5 microM PGF2 alpha in large but not small cells. These data suggest that PGF2 alpha releases calcium from a TG-sensitive intracellular calcium pool in ovine large luteal cells.

[Changes in the distribution of muscle fiber types in the M. longissimus dorsi of boars during growth, prepared by different histochemical methods]. / Veränderungen der Muskelfasertypenverteilung im M. longissimus dorsi von Ebern während des Wachstums, dargestellt mit verschiedenen histochemischen Methoden.

Samples of the M. longissimus dorsi from growing up pigs were taken by a shooting-biopsy. The types of muscle-fibers could be demonstrated by two different histochemical staining procedures. With these two histochemical staining procedures significant differences between the different structure of the muscle-fibers during increasing age of the pigs could be detected.

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion.

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: I. Alterations in cytosolic-free calcium levels and calcium flux.

The effect of prostaglandin F2 alpha (PGF2 alpha) on cytosolic calcium homeostasis was studied in suspensions of ovine large or small luteal cells from superovulated ewes. In large cells loaded with fura-2 (AM), resting cytosolic-free calcium ([Ca2+]i) was 62 +/- 5 nM (Hanks' medium, pH 7.15), and PGF2 alpha (0.5 microM) induced a rapid transient increase in [Ca2+]i to 152 +/- 6 nM, which then decreased to 97 +/- 6 nM within 3 min and remained at this level for the remainder of the treatment period (10-20 min). PGF2 alpha did not alter intracellular pH (pHi) in cells loaded with snarf-1 (AM) (pHi indicator). The transient nature of the [Ca2+]i increase was due, at least in part, to the ability of PGF2 alpha to stimulate (P less than 0.05) 45Ca2+ efflux. In small cells, resting [Ca2+]i was 57 +/- 5 nM, and no change in [Ca2+]i levels or pHi occurred with the addition of PGF2 alpha. PGF2 alpha also did not affect 45Ca2+ efflux in small cells. Calcium uptake was not significantly altered by PGF2 alpha in large or small cells. Data from kinetic analysis of the calcium transient was best fit to a two-compartment model consisting of a rapidly effluxing compartment and a slowly effluxing compartment. The size and rate constants were 62 +/- 10 nM and 3.6 +/- 1 min-1, respectively, for the rapidly effluxing compartment and 140 +/- 9 nM and 0.02 +/- 0.002 min-1, respectively, for the slowly effluxing compartment. These results provide evidence for a direct effect of PGF2 alpha specifically on the ovine large luteal cell that involves alterations in [Ca2+]i and calcium flux. This effect is likely to be involved in intracellular mediation of the signal for luteal regression.

[Formaldehyde sediment in incubators following disinfection]. / Formaldehydrückstände in Inkubatoren nach der Desinfektion.

Measurements in incubators revealed the presence of formaldehyde concentrations involving a health risk for premature and normal newborns kept and cared for in incubators. Prior to measurements, the incubators had been disinfected by means of formaldehyde vapours in an "Aseptor" disinfecting cabinet (Drägerwerk AG, Lübeck) and then ventilated in strict adherence to operating instructions. The elevated formaldehyde concentrations found had been due to residues of paraformaldehyde and urotropin on the surfaces of the disinfected apparatus, liberating formaldehyde by hydrolysis depending on temperature and relative humidity. There should be a basic reconsideration of the present practice of incubator disinfection. From experiments with activated-carbon filters in incubators it would seem that there is a chance of reducing such formaldehyde concentrations.