Low Molecular Weight Bands Research Articles

Zoledronate acid modifies the lipid transporters profile in human prostate cancer cell lines

21135 Background: The mechanism through wich zoledronic acid exerts its activity is poorly understood. Low density lipoprotein receptor (LDLR) and scavenger receptor class B type 1 (SRBI) overproduction is an important mechanism in cancer cells for obtaining more essential fatty acids and cell growth. The effects of in vitro zoledronate treatment on the lipid metabolism of prostate cancer cell lines were studied. Methods: Three prostatic cancer cell lines, androgen insensitive PC3, androgen dependent low-passage (LP) LNCaP and androgen independent high-passage (HP) LNCaP cells were studied. Cells were plated either in RPMI with 5% foetal calf serum and lipoprotein depleted serum (LPDS) and were treated with zoledronate at different concentrations. The lipid transporters profile was analyzed by western blotting for LDLR and SRB1. Results: The same LDLR bands profile was observed in all cell lines, 160 and 105 kDa. The basal levels of LDLR were higher in the PC3 cells. Zoledronate therapy induced LDLR expression in all cell lines but PC3 were less sensitive to this effect. Cells cultured with LPDS showed an enhanced expression of LDLR and PC3 cells were less sensitive to this effect. HP LNCaP cells were the most affected by lipoprotein deprivation however this effect diminished 72 hours after treatment. The bands profile for SRBI consisted of a 65 kDa predominant band and a 40 kDa band in both LP/HP LNCaP cells. In PC3 cells main band was located in 65 kDa and accessory band in 30kDa. The basal levels of the 65kDa band were higher in HP than in LP LNCaP or PC3 cells and zoledronate therapy caused a dose- dependent induction in HP LNCaP and dose-dependent reduction in PC3 cells, LP LNCaP cells were resistant to the treatment. LPDS induced SRBI levels in all cell lines inverting the effect caused by zoledronate in HP LNCaP cells in complete culture medium and at high doses (100μM) a complete inhibition of SRBI protein was found. Low molecular weight bands changed in the same way as the 65 kDa band. Conclusions: LDL-R and SRBI have been isolated in prostate cancer cell lines. Based on previous cell growth studies, the lipid transporters profile might be significantly involved in the resistance to zoledronate therapy. Lipoprotein regulation pathways should be considered in the therapy of metastatic prostate cancer. No significant financial relationships to disclose.

MDM2 Binding Induces a Conformational Change in p53 That Is Opposed by Heat-shock Protein 90 and Precedes p53 Proteasomal Degradation

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.

Heparin Binding Induces a Conformational Change in Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.

Helicobacter pylori-associated chronic idiopathic thrombocytopenic purpura and low molecular weight H. pylori proteins

We examined the association between Helicobacter pylori infection and chronic idiopathic thrombocytopenic purpura (cITP) to clarify the development of H. pylori-associated cITP. cITP patients were classified into 3 different groups: Hp-negative (HP-N); Hp-positive, completely or partially responsive to treatment (CR); and Hp-positive and unresponsive to treatment (NR). Reactivity of antibodies to H. pylori before and after eradication was examined by immunoblotting. We used immunoblotting with immunoprecipitation to establish whether platelets form complexes with H. pylori proteins and if these complexes react with patients' sera. CR group showed large (>50 kDa) and low molecular weight protein bands, especially of 36, 27 and 17-kDa. These low molecular weight bands were detected significantly more in the CR group compared to other groups. When healthy human platelets were incubated with H. pylori lysate, they aggregated with the lysate, indicating that complexes were formed between the platelets and the lysate. The complexes immunoprecipitated with anti-human thrombocyte antibodies, and showed a 17 kDa band in the CR, but not in other groups. At least 3 low molecular weight proteins of H. pylori were involved in H. pylori-associated cITP. Immunocomplexes consisting of platelets, low molecular weight proteins of H. pylori and anti-H. pylori antibodies may represent an extra mechanism in development of H. pylori- associated cITP.

On resolving the molecular identity of the endothelial cell nucleosome assembly protein

Nucleosome assembly protein (NAP) is a chromatin-associated protein that fulfills an important role in cell memory, and has been incriminated in the control of cell growth. We have recently shown that NAP expression is an important determinant of the endothelial vasculogenic phenotype. Western analysis using a monoclonal antibody specifically targeting the 4a8 epitope of NAP has consistently resolved two bands. The higher molecular weight band is 58 kD, and corresponds to full length NAP. The lower molecular weight band is 53 kD, and has been postulated to be a degradation product, although this lower band has yet to be specifically identified. To resolve the molecular identity of both NAP bands, full-length NAP with a hemaglutinin tag was expressed in pulmonary artery endothelial cells (PAEC). Western analysis of PAEC nuclear and whole cells lysates using a hemaglutinin antibody resolved only a 58 kD band, indicating the 53 kD product is not strictly a degradation product. Short hairpin RNA (shRNA) was then utilized to knockdown NAP expression in pulmonary microvascular endothelial cells. This knockdown strategy resulted in disappearance of the 53 kD band in nuclear extracts, and decreased the 58 kD band. These data suggest that full-length NAP encodes for the 58 kD product, and while the 53 kD product is related to NAP, it may be derived through either differential processing of the full-length mRNA product, or result from expression of a related gene. Supported by:HL66299.

Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway

Isolates of atypical scrapie recently identified in sheep and goats in France were compared with Nor98 isolates reported in Norway. Western blot methods for characterization of the protease-resistant prion protein showed that all these isolates shared a unique biochemical signature: 5 groups of bands, including a characteristic band of apparent low molecular weight (11 kDa). This pattern could originate from the presence of 3 different protease cleavage products, including the 11 kDa most likely cleaved at both N- and C-sides of the protein. Genetic data, which strongly suggested the higher susceptibility of AHQ and AF141RQ animals in French cases, resembled earlier data from Nor98 scrapie.

Negative Regulation of Osteoclastogenesis by Ectodomain Shedding of Receptor Activator of NF-κB Ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

Washout of heme-containing proteins dramatically improves tetrazolium-based infarct staining

Introduction Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. Methods Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 °C for up to 3 weeks. Results Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INF. However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. Discussion These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury.

Low molecular weight species of tau in Alzheimer's disease are dependent on tau phosphorylation sites but not on delayed post-mortem delay in tissue processing

Gel electrophoresis and Western blotting of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau in Alzheimer disease (AD) have been used to analyze the pattern of phospho-tau by using different antibodies directed to the amino-terminal, core and carboxyl terminus of tau, and by using samples with increased artificial post-mortem delay in order to gain understanding on the characteristics of the band pattern and its vulnerability to post-mortem degradation. In addition to the typical profile of three major bands of 68, 64 and 60 kDa, several bands of lower molecular weight have been distinguished in frontal cortex homogenates in four AD cases stage V of Braak and Braak in optimal samples with 2 h of post-mortem delay. Lower bands, ranging from 60 to 22 kDa, are best seen with antibodies directed to the core of tau protein and, particularly, to the carboxy-terminus, thus suggesting the presence of truncated or cleaved forms of tau containing the C-terminal region. This pattern is not the result of post-mortem degradation, as artificial post-mortem delay of the same sample does not reveal the appearance of new bands with time. On the contrary, tau degradation, manifested as a reduction in the number and intensity of the bands, may occur between 8 and 26 h post-mortem and is universal in samples with post-mortem delays of 50 h.

The α IIbG236E Mutations Causes Glanzmann Thrombasthenia by Impairing Association with β3.

Glanzmann Thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3. The recent solution of the crystal structure of αIIbβ3 revealed that the N-terminal domain of αIIb is folded in a complex structure, a β-propeller formed by seven blades. The role of the β-propeller is to bind fibrinogen and to associate to β3. In the endoplasmic reticulum pro-αIIb associates with β3, it is then transferred to the Golgi, where it is cleaved in two chains disulfide bonded (mature αIIb); mutations that impair the association of pro-αIIb to β3 are able to cause GT. The architecture of the propeller is sustained by several prolines that stabilize the entire structure. From molecular modeling studies it had been postulated that the residue spatially adjacent to proline 258 can only be a glycine because there is no physical space for other residues. Analyzing the GT mutations of Italian patients recently described by D'Andrea et al. (Thromb Haemost., 2002) we found that a patient with type I GT had a αIIb G236E missense substitution, hence we decided to verify if the substitution of glycine 236 was hampering αIIb β3 association. The G236E mutation was introduced by site directed mutagenesis in pCDNA3.1 containing normal human αIIb cDNA. Human embryonic kidney (HEK) cells were transfected either with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. By flow cytometry analysis the percentage of HEK cells transfected with αIIbG236Eβ3 that reacted with 10E5-FITC (anti αIIbβ3) was 7±1% while the percentage of cells transfected with αIIbβ3 that reacted with 10E5-FITC was 37±13%. When the binding of an antibody directed against β3 (VIPL-2-FITC) was evaluated, we observed that cells expressing αIIbG236Eβ3 did bind the antibody, indicating that the normal transfected β3 was associating with the endogenous αV. HEK cells transfected with either αIIbβ3 or αIIbG236Eβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. Both lysates were reacting at the same level with antibodies directed against β3. When immunoblotting was done in non-reducing conditions utilizing an antibody reacting with αIIb (mab1990) a band corresponding to αIIb was present in both lysates, although less intense in cells transfected with αIIbG236Eβ3. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbG236Eβ3 the ratio pro-αIIb: αIIb was 1:1. Moreover, when tested against lysates of cells transfected with αIIbG236Eβ3, the antibody against αIIb recognized multiple bands of lower molecular weight, presumably degradation products of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against αIIb or αIIbβ3 and immunoblotted with an antibody reacting with β3. While in immunoblots from cells transfected with αIIbβ3 a band corresponding to β3 was strongly detectable, in immunoblots originating from cells transfected with αIIbG236Eβ3 a very weak band at the same level of normal β3 was detected only in immunoprecipitates using 10E5, a complex dependent antibody. In conclusion we demonstrated that αIIbG236E is a mutation that causes GT by impairing the association with β3 in the endoplasmic reticulum; the effect of the mutation is likely to create a severe misfold of the β-propeller and the molecular modeling prediction of the need of a glycine at position 236 is probably confirmed.

Immunoblot analysis of specific antigen bands predictable for Dirofilaria immitis infection in cats

Serial sera from four mongrel cats experimentally inoculated with infectious larvae of Dirofilaria immitis were analyzed by immunoblot patterns against a phosphate buffered saline-extract of D. immitis. Antigen-specific protein bands detected indicate that the low molecular weight bands of 36, 32, 22, 19 and 14 kDa, are predictable for positive adult worm infection, suggesting diagnostic usefulness for adult D. immitis infection in cats.

Influence of Thermal Processing on the Allergenicity of Peanut Proteins

Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins. The aim of this study was to assess the effect of thermal processing on the IgE-binding capacity of whole peanut protein extracts and of the major peanut allergens Ara h 1 and Ara h 2. Whole proteins, Ara h 1, and Ara h 2 were extracted and purified from raw, roasted and boiled peanuts using selective precipitation and multiple chromatographic steps, and were then characterized by electrophoresis and mass spectrometry. The immunoreactivity of whole peanut extracts and purified proteins was analyzed by the enzyme allergosorbent test (EAST) and EAST inhibition using the sera of 37 peanut-allergic patients. The composition of the whole protein extracts was modified after heat processing, especially after boiling. The electrophoretic pattern showed protein bands of low molecular weight that were less marked in boiled than in raw and roasted peanuts. The same low-molecular-weight proteins were found in the cooking water of peanuts. Whole peanut protein extracts obtained after the different processes were all recognized by the IgE of the 37 patients. The IgE-binding capacity of the whole peanut protein extracts prepared from boiled peanuts was 2-fold lower than that of the extracts prepared from raw and roasted peanuts. No significant difference was observed between protein extracts from raw and roasted peanuts. It is noteworthy that the proteins present in the cooking water were also recognized by the IgE of peanut-allergic patients. IgE immunoreactivity of purified Ara h 1 and Ara h 2 prepared from roasted peanuts was higher than that of their counterparts prepared from raw and boiled peanuts. The IgE-binding capacity of purified Ara h 1 and Ara h 2 was altered by heat treatment and in particular was increased by roasting. However, no significant difference in IgE immunoreactivity was observed between whole protein extracts from raw and roasted peanuts. The decrease in allergenicity of boiled peanuts results mainly from a transfer of low-molecular-weight allergens into the water during cooking.

Effect of Calcium Chloride Marination on Electrophoretical and Structural Characteristics of Beef, Horse, Rabbit and Chicken Meat

Marinating with calcium chloride had been employed to activate the endogenous proteolitic enzymatic system in muscle tissue. The activation of calpains leads to the disruption of major proteins responsible for myofilament structure. Nonetheless, intrinsic differences regarding enzymatic activity between different species need to be established, which concerns protein degradation and ultrastructural changes. In this study, four species—horse, chicken, rabbit and beef—were marinated in 150 mM CaCl2 solution. Calpain activity was determined by low and high molecular weight protein degradations (SDS-PAGE) and light microscopy, which was compared to a non-treated control. All the studied species presented an increase in the number of low molecular weight bands, corresponding to the 30 kDa polypeptide that resulted from tropomiosin degradation. High molecular weight protein degradation corresponds to the formation of breakdowns and amorphous regions in tissue micrographs. These results support the evidence of meat quality improvement by calcium marination.

Antisense-Induced Fas mRNA Degradation Produces Site-Specific Stable 3'-mRNA Fragment by Exonuclease Cleavage at the Complementary Sequence

Antisense-mediated degradation of target mRNA is achieved by the enzymatic action of nuclease RNase H. The enzyme recognizes hybrid RNA-DNA duplexes and hydrolyzes the RNA strand. Here, we compared six different phosphorothioate oligonucleotides for their ability to induce target-specific mRNA degradation in cultured mouse AML12 cells. We targeted transcripts of the cell surface receptor Fas and analyzed the levels of mRNA by Northern blotting and ribonuclease protection assay (RPA). Four of the tested antisense oligonucleotides reduced the mRNA levels significantly. Cultures treated with one of the antisense molecules resulted in a shifted band on Northern blots. This band of lower molecular weight was not detected after 6 hours of transfection but appeared at 24 hours. By RPA, the product was shown to be a 3'-cleavage fragment of the full-length Fas mRNA. The RPA also mapped the stable fragment to start within the antisense complementary sequence.

Detection of nascent and/or mature forms of oviductin in the female reproductive tract and post-ovulatory oocytes by use of a polyclonal antibody against recombinant hamster oviductin.

Oviductins belong to a family of glycoproteins that have been suggested to play several roles during the early processes of reproduction. Recently, a polyclonal antibody was raised against recombinant hamster oviductin (rhaOv(m)). Here the anti-rhaOv(m) antibody was used to investigate the sites of localization of oviductin in the female golden hamster. In the hamster oviduct, immunolabeling was restricted to the content of the Golgi saccules and secretory granules of the non-ciliated oviduct cells. After its release into the lumen, oviductin becomes associated with the zona pellucida of post-ovulatory oocytes. In unfertilized oocytes, oviductin was also detected in membrane invaginations along the oolemma and in some vesicles within the ooplasm. Furthermore, oviductin was detected over the microvilli and within multivesicular bodies of uterine epithelial cells. Western blotting analysis revealed the presence of oviductin in the hamster oviduct but not in the uterus or ovary. In the oviduct, the anti-rhaOv(m) antibody detected a polydispersed band corresponding to native oviductin (160-350 kD) and several lower molecular weight bands (<100 kD) corresponding to nascent and partially glycosylated forms of oviductin. The anti-rhaOv(m) antibody provides an additional tool for investigation into the cytochemical and biochemical properties of different forms of hamster oviductin in the female reproductive tract.

Presence of estrogen receptor (ER)-like proteins and endogenous ligands for ER in solanaceae

The synthesis of steroid hormones in different plant species and the possibility that these molecules could regulate cell growth, tissue differentiation and germination has been reported. However, the mechanism of action of these endogenous steroids in plant cells is still poorly understood. In the present work, binding experiments with [3H]17β-estradiol in the presence and absence of an excess of unlabeled 17β-estradiol showed that Solanum glaucophyllum and Lycopersicon esculentum organs contain estrogen-binding sites. Scatchard analysis detected saturable [3H]17β-estradiol-binding sites (Kd∼6.6nM; Bmax∼1140fmol/mg protein) in S. glaucophyllum callus tissue. Estrogen-like compounds were detected in lipid extracts from S. glaucophyllum and L. esculentum. Moreover, the lipid fraction was able to compete with [3H]17β-estradiol for binding to estrogen receptor (ER) from breast cancer MCF-7 cells as well as with estrogen-binding sites present in both plant species. Western blot analysis with monoclonal antibodies against different domains of the ER α, detected a ∼67kDa band in various organs of both plant species. These proteins were able to bind estradiol in ligand blot assays using 17β-estradiol macromolecular derivatives as ligands. Western and ligand blot experiments of subcellular fractions from callus tissues of S. glaucophyllum showed that the ER-like protein of ∼67kDa was most concentrated in the nuclear fraction. Reactive bands of lower molecular weight were also detected. Altogether these results provide evidence about the existence of estrogen-binding proteins and endogenous ligands in Solanaceae.

Activation of metalloproteinases in systemic conduit arteries after myocardial ischemia

Recent data from our laboratory showed that metalloproteinases (MMPs) and Tissue inhibitor of MMPs (TIMPs) are activated very early after myocardial ischemia. Hemodynamic changes due to myocardial ischemia appear to play a significant role in myocardial MMPs/TIMPs activation. Since the peripheral vasculature is subjected to the same alterations in hemodynamics as the myocardium, we hypothesized that myocardial ischemia and infarction activates peripheral vascular MMPs/TIMPs in a time-dependent manner. MMPs activation was measured in aortic tissue homogenates using gelatin zymography and their abundance was measured using immunoblot analysis. Activation of TIMPs was measured using reverse zymography. MMP activities after ischemia were compared to nonischemic tissue. ProMMP-9 activities were decreased (P 0.05) by 52, 86, 51% at 1 min, 5 min, and 7 days after ischemia, respectively. ProMMP-2 activities were decreased (P 0.05) by 33, 78, 38% at 1 min, 5 min, and 7 days after ischemia, respectively. MMP-2 activities were decreased (P 0.07) by 19% and 17% at 1 min and 5 min after ischemia, respectively. In contrast, MMP-2 activities were increased (P 0.05) by 40% at 7 days after ischemia. ProMMP-9, ProMMP-2 and MMP-2 activities were absent at 21 days after ischemia (Fig.1). Western blot analysis suggests that MMP-2, MMP-9, and MMP-13 may be activated five minutes after ischemia, evident by the disappearance of the proenzymes (high molecular weight bands) and the appearance of the active enzymes (low molecular weight bands) in the ischemic tissue compared to nonischemic vessels (Fig. 2). In addition, TIMP-1 and TIMP-2 activities were decreased (P 0.05) at five minutes, 7 and 21 days after ischemia. Thus, myocardial ischemia modulates the MMPs/TIMPs balance early after ischemia in the peripheral conduit arteries.

Effect of dietary protein on calpastatin in canine skeletal muscle.

The cysteine proteinases, mu- and m-calpain, along with their inhibitor, calpastatin, have been hypothesized to play a role in skeletal muscle protein degradation. Because nutrition has previously been shown to influence the expression of calpastatin, the working hypothesis of this study was that the quantity and source of dietary protein could influence regulation of the calpain system in muscle. The objectives to support this hypothesis were to determine the effects of dietary protein (amount and source) on the expression of calpastatin in canine skeletal muscle. This study comprised eight diets with seven dogs per diet. A biopsy was taken from the biceps femoris of all 56 dogs before and after 10 wk on their respective diets. This experimental design allowed examination of change within individual dogs. Diets 1 to 4 contained 12% total protein derived from chicken and/or corn gluten meal in ratios of 100:0, 67:33, 33:67, and 0:100%, respectively. Diets 5 to 8 contained 28% total protein with protein sources and ratios identical to Diets 1 to 4. Differences in calpastatin were examined qualitatively using SDS-PAGE and immunoblotting, and semiquantitatively with densitometric analyses. The majority of the calpastatin blots showed three distinct calpastatin bands, the uppermost appearing at approximately 110 kDa. Diet 5 (28% CP, 100% chicken) resulted in an increase in the expression of the 110-kDa calpastatin band compared with the other two lower molecular weight bands in the same samples. Muscle from dogs fed Diet 5 showed greater increase in (P < 0.05) calpastatin intensity of the topmost band than those fed Diet 8 (0:100; chicken:corn gluten meal). Diet 5 (100:0; chicken:corn gluten meal) showed greater total calpastatin intensity than Diet 8 (0:100; chicken:corn gluten meal). These data suggest that dogs fed a diet containing a higher total percentage of chicken protein may have a greater potential to regulate calpain-mediated degradation of muscle protein than dogs fed diets containing corn gluten meal.

Selective up-regulation of tumor necrosis factor receptor I in tumor-bearing rats with cancer-related cachexia

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) are important mediators in cancer cachexia; however, the expression of these cytokines and their receptors in tumor-bearing animals is poorly characterized. We analyzed expression of TNF-alpha, IL-6, tumor necrosis factor (TNF-RI, TNF-RII) and interleukin 6 (IL-6R) receptors in the brain, kidney, spleen, liver, muscle, ascite tumors and serum, from Yoshida AH-130 hepatoma-bearing rats. TNF-alpha increased in the brain, spleen, liver, and muscle of cachectic animals; IL-6 increased in the liver and muscle. AH-130 cells expressed a good level of TNF-alpha; on the contrary, no TNF-alpha or IL-6 protein was detected in the serum of either tumor-bearing or control animals. TNF-RI mRNA was up-regulated in the spleen, liver and muscle of tumor-bearing rats. TNF-RI protein levels confirmed up-regulation in the spleen and liver, but failed to detect any increase in the muscle. Western blotting against TNF-RI revealed two bands of lower molecular weight in cachectic muscle, suggesting proteolysis involving TNF-RI. No significant increase of either TNF-RII or IL-6R was observed. This is the first demonstration of a selective up-regulation of TNF-RI in cancer cachexia and suggests that local production of TNF-alpha and IL-6 is a corner-stone in the induction/maintenance of this syndrome.