Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues

A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25 degrees C) which is known to methylate and acetylate--respectively--free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30 degrees C) eliminated the UA staining without affecting that due to PTA. It is concluded that--COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.     

Cytochemical studies on RNP complexes produced by puff 2-48BC in Drosophila hydei

Differential staining of the core and RNP particles of RNP complexes in puff 2–48 BC in salivary gland chromosomes of Drosophila hydei was achieved with aqueous uranyl acetate (UA) at low pH, with UA in acetone, with phosphotungstic acid (PTA) in organic solvents, and with aqueous PTA at pH 5 and 6. A comparison of the results of UA and PTA staining under various conditions indicate that the proteins in the core region and in the RNP particles connected to it differ with respect to their amino-acid composition (arginine and lysine residues). — The staining mechanism of PTA and UA is discussed.     

Mechanisms of chromation hematoxylin stains

Summary In chromation hematoxylin sequence stains of Weigert-Smith-Dietrich type an exploration is presented of the nature of chromium binding tissue end groups, of the valency of the bound chromium and of the mechanisms and conditions of its binding.At 2–4° C prolonged (2–8 weeks) mordanting in 2.5% K₂Cr₂O₇ at pH 3.5 engenders staining with acetic hematoxylin essentially limited to histochemical ethylenic sites, completely preventable by prior aqueous bromination and unaffected by sulfation or acetylation. Erythrocytes, myelin and bile casts are examples of reactive tissue elements. At 24° C and more so at 60° C there is increased reaction of muscle, cytoplasm, nuclei and other structures; the reaction is now partially blocked by acylations and is only partly prevented by bromination, indicating participation of hydroxyl groups. Deamination decreases reaction at 60° C of protein background, but not notably of myelin, erythrocytes, or bile casts. The previously reported carboxyl binding of hot chromation oxyphilia is almost inapparent when chromation is done at 3° C. Chromic acid is less selective in its action, producing some background staining even at 3° C; K₂CrO₄ engenders no acetic or neutral hematoxylin staining, even at 6.6% and 24 hour mordanting at 24° or 60° C.Chromium deposited from dichromate or chromic acid mordanting reacts with hematoxylin solutions at pH 2.5 to 7.0, that deposited from Cr III salts reacts only at pH 6–7. Mordanting with Fe III, Fe II, Cu II and Sn II salts yields hematoxylin staining respectively from pH 2.5, 4.0, 5.0 and 2.5 upward. After K₂Cr₂O₇ mordanting brief reductions and acid treatments restrict hematoxylin staining to the same neutral pH zone as that produced after Cr III mordanting, but the pH 7 staining capacity of Cr deposited from K₂Cr₂O₇ is more resistant to extraction agents than that from Cr III solutions. It is therefore concluded that the chromium deposited in dichromate mordanting is of a higher valency than Cr III and it is suggested that Cr IV is present and bound to double bond sites in ring esters in the same manner as has been formulated for Mn and Os in the attack of KMnO₄ and OsO₄ on ethylene double bonds.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health, Bethesda, Maryland. Presented in part at the Third International Congress for Histochemistry and Cytochemistry, New York 1968.     

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

The dihydropyridine receptor (DHPR) β_1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β_1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β_1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β_1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β_1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β_1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β_1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β_1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β_1a and RyR1 in resting myotubes.     

Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension in mice

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 mA intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p < 0.01 in EA and MA) and MuRF1 (p < 0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p < 0.05 in MA) and TRPV4 (p < 0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation.     

Asymmetric catalysis of the Strecker amino acid synthesis by a cyclic dipeptide

Summary A novel cyclic dipeptide —cyclo[(S)-His-(S)-NorArg] — has been prepared which catalyzes an enantioselective version of the Strecker amino acid synthesis. The catalyst, when present in 2 mol % quantity in methanol solution, catalyzes the addition of hydrogen cyanide toN-alkylimines to afford-amino nitriles in high yield and high enantiomeric excess. Furthermore, acid hydrolysis ofN-benzhydryl--amino nitriles afforded the corresponding-amino acids directly. This methodology affords a variety of arylglycines in exceptionally high enantiomeric excess, but aliphatic amino acids were obtained with low enantioselectivity. Current efforts are underway to expand the scope of this reaction, as well as to elucidate the mechanism of catalysis and the roles played by substrate and catalyst in determining the stereochemical outcome of the reaction.A preliminary communication on this work has recently appeared in: Iyer MS, Gigstad KM, Namdev ND, Lipton MA (1996) J Am Chem Soc 118: 4910–4911.     

Psophocarpus tetragonolobus agglutinin reveals N-acetyl galactosaminyl residues confined to endothelial cells and some epithelial cells in human tissues

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

AAMP, a Newly Identified Protein, Shares a Common Epitope with α-Actinin and a Fast Skeletal Muscle Fiber Protein

AAMP (angio-associated migratory cell protein) shares a common epitope with α-actinin and a fast-twitch skeletal muscle fiber protein. An antigenic peptide, P189, derived from the sequence of AAMP was synthesized. Polyclonal antibodies generated to P189 readily react with AAMP (52 kDa) in brain and activated T lymphocyte lysates, α-actinin (100 kDa) in all tissues tested, and a 23-kDa protein in skeletal muscle lysates. The antibody's reactivity for α-actinin can be competed with the purified protein. Activation of T lymphocytes does not alter the degree of α-actinin reactivity with anti-P189 as it does for AAMP's reactivity in these lysates. Competition studies with peptide variants show that six amino acid residues, ESESES, constitute a common epitope in all three proteins in human tissues. The antigenic determinant is continuous in AAMP but discontinuous (or assembled) in α-actinin. α-Actinin does not contain this epitope in its linear sequence so reactivity is attributed to an epitope formed by its secondary structure. Limited digestion of the reactive proteins with thermolysin destroys anti-P189’s reactivity for α-actinin while reactivity for recombinant AAMP is retained. Specificity of anti-P189 for human skeletal muscle fast fibers seen on immunoperoxidase staining may be explained by anti-P189’s reactivity with a 23-kDa protein found only in skeletal muscle lysates. Its pattern of reactivity is the same as that obtained using monoclonal anti-skeletal muscle myosin heavy chain in type II (fast-twitch) fibers.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

The rotifer fauna and population dynamics of Lake Studer 2 (Kerguelen Archipelago) with description ofFilinia terminalis kergueleniensis n. ssp. and a new record ofKeratella sancta Russel 1944

Two species of Anabas Cuvier, 1816 (Osteichthyes: Anabantidae): A. testudineus (Bloch, 1795) and A. oligolepis Bleeker, 1855 are widely distributed in India. Since both species are represented in one habitat — Lake Kolleru, and are very closely related, they were compared using starch gel electrophoresis. Electrophoretic separation of proteins of eye lens, skeletal muscle and heart muscle revealed differences such as i) absence of one protein fraction in one of them, ii) mobility and iii) staining intensity of some of the bands. The esterase (non-specific) patterns of serum, liver, kidney, skeletal muscle, heart muscle and eggs also showed differences. The LDH fraction of eye lens showed a difference in mobility.     

Biochemical differentiation of plastids and other organelles in rye leaves with a high-temperature-induced deficiency of plastid ribosomes

Summary 1. In developing rye (Secale cereale L.) leaves the formation of plastidic ribosomes was selectively prevented in light as well as in darkness, when the seedlings were grown at an elevated temperature of 32° instead of 22° where normal development ocurred. Plastid ribosome deficient parts of lightgrown leaves were chlorotic at 32°. — 2. At both temperatures the leaves contained under all conditions (light or dark, on H₂O or nutrient solution) equal or very similar amounts of total amino nitrogen. In light, the contents of total protein and dry weight were lower at 32° than at 22°, especially when the plants were grown on nutrient solution. — 3. Mitochondrial marker enzymes had normal or even higher activities in 32°-grown leaves. Respiration rates were similar for segments of leaves grown on water in light either at 32° or at 22° but by 20–30% lower for 32°-grown plants when they had been raised in darkness or on nutrient solution. In contrast to 22°-grown tissue, respiration of 32°-grown leaf segments was rather insensitive to KCN. Comparative inhibitor studies indicated the presence of both the cyanide-sensitive and the cyanide-insensitive pathway of respiration in 32°-grown leaves. — 4. Leaf microbody marker enzymes were present in leaves grown at 32°. From chlorotic parts of 32°-light-grown leaves a typical microbody fraction was isolated on sucrose densitygradients. — 5. Leaves of seedlings grown at 32° contained only very low levels of ribulosediphosphate carboxylase activity and of fraction I protein. Photosynthetic ¹⁴CO₂-fixation of such leaves was only a few per cent of that observed in normal leaves, and no photosynthetic oxygen evolution was observed in chlorotic leaf segments. However, ten other soluble enzymes which are exclusively or partially localized in chloroplasts reached high activities under all conditions at 32° (Table 4). — 6. From chlorotic parts of 32°-light-grown leaves as well as from etiolated 32°-grown leaves a fraction of intact plastids was isolated and purified by sucrose gradient centrifugation which contained several soluble chloroplast enzymes. From the results we conclude that cytoplasmic protein synthesis must contribute a functional chloroplast envelope including the mechanism for the recognition and uptake of chloroplast proteins which are synthesized on cytoplasmic ribosomes.     

On the specificity of staining by Alcian blue in the study of human,foetal adenohypophysis

Summary The specificity of Alcian blue staining has been investigated on a material comprising the adenohypophysis from 26 human foetuses.Two distinct types of alcianophilic cells were observed. The first cell type appears about 28 mm CRL forming small groups at the epithelial-mesenchymal junction and beneath the anlage of the fibrous capsule. Besides its alcianophilic property the cell shows a pronounced maltase-resistant PAS-positivity. The alcianophilia is due to carboxylgroups — probably of proteins as no sialic acid could be detected. By the performic acid — Alcian blue method the cell seems to show a high content of SH and S-S groups.The second type of cell appears about 70 mm CRL and is mostly located singly. It shows a pronounced alcianophilia — probably due to sulphate groups and to some extent to carboxyl groups.The role of the alcianophilic properties of the cells in typing definite cells of the foetal adenohypophysis was discussed. — Finally the pitfalls caused by different fixatives and by the use of the general polychrome staining methods for the typing of pituitary parenchymal cells were discussed.This work was supported by grants from the Association for the Aid of Crippled Children, New York, and from the Danish State Research Foundation, Copenhagen.     

Morphochemical analysis of mucous cells in the skin and slime glands of hagfishes

Summary The mucous cells of the epidermis and slime glands in three species of Pacific hagfishes were studied histochemically to determine the presence or absence of acidic and neutral mucosubstances. Most mucous cells contained diastase-resistant PAS reactivity that varied in intensity. A few mucous cells contained diastase-labile substances exclusively.Acidic groups were detectable due to their stainability by several basic dyes which were utilized singly or in combination. Considerable species diversity of hagfish mucins was noted in their affinity toward azure A at pH 1.0, 2.0, and 3.0 depending on whether sections were viewed wet from the staining jar or viewed after dehydration and mounting. At pH 1.0, a few mucous cells were metachromatic under both conditions while the majority were unstained in both types of section. As the pH was raised, the majority of cells were metachromatic when viewed wet or mounted. Most mucous cells were reactive toward alcian blue at pH 1.0, aldehyde fuchsin and the high iron diamine reagents. Each of these reactions was absent following exposure of sections to acidic (0.1N HCl) methanol for 4 hours at 60°C while control sections exposed to unacidified methanol or acidified isopropanol for the same time and temperature resulted in no loss of staining.Sialidase-labile acidic groups were detected in the epidermal mucous cells of one species of hagfish. Following prior treatment of sections with Vibrio cholerae sialidase for 16–24 hours at 39° C, there was a reduction of metachromasia of the mucous cells produced by azure A at pH 3.0. Although confirmatory autoradiographic and biochemical data on hagfish mucosubstances are lacking, the histochemical staining results and their subsequent modifications by enzyme and chemical treatment indicate that the majority of these mucins contain sulfomucin while a few are composed of sialomucin. Similar results of previous histochemical and autoradiographic studies of epithelial secretions from higher animals correlated, in some instances, with the biochemical data for those mucins.Supported by Research Grant AM — 11064 of the United States Public Health Service.Recipent of a Lederle Medical Faculty Award, 1968–1971.     

Subcellular immunolocalisation of fatty acid translocase (FAT)/CD36 in human type-1 and type-2 skeletal muscle fibres

Limited information exists about the putative role and expression in human skeletal muscle cells of the 88-kDa integral membrane protein fatty acid translocase (FAT), highly homologous to the human leucocyte differentiation factor CD36. Therefore, we investigated in healthy male individuals the muscle (m. vastus lateralis) fibre type specific expression and subcellular localisation of FAT/CD36. For this purpose four different monoclonal antibodies raised against human and mouse FAT/CD36 were used. Acetone or methanol/acetone fixation were tested. Serial cryosections were cut at –20°C, thaw-mounted on uncoated glass slides and air-dried before processing indirect immunofluorescence assays. Images were examined in a Nikon ER800 microscope, digitally captured, processed and analysed by LUCIA laboratory software. Three antibodies showed that FAT/CD36 was: (1) most abundantly expressed in capillary endothelium, (2) colocalised with caveolin-3, which indicates that FAT/CD36 is in the sarcolemma, or its close vicinity, and (3) abundantly expressed in (or in the close vicinity) of the sarcolemma and intracellular structures of type-1 muscle fibres, and much less abundantly in the sarcolemma of type-2 muscle fibres. One of the antibodies raised against mouse CD36 also detected myosin heavy chain 1, which makes it unsuitable in skeletal muscle research. The fixation (acetone or methanol/acetone) was found to be highly important for the result.     

Hot chromation oxyphilia

Summary On exposure of formol or methanol chloroform fixed sections to solutions of 2.5–5% potassium dichromate or 4.2% chromic acetate for 18 hours at 60° C a strong relative oxyphilia develops. As measured with buffered solutions of azure A eosin B the point at which various tissue elements pass from red to blue staining is elevated by 3 to 4 pH units. A similar change occurs in K₂Cr₂O₇ in about 21 days at 24° C and at 4° C only a slight elevation of protein pK appears in 6 weeks. Ferric chloride and ferrous sulfate solutions of comparable metal content produce a less marked shift (about 2 pH units) and relatively slight similar action is produced by aluminium and copper salts (about 1 pH unit). Heating in water, NaCl, KH₂PO₄ and K₂CrO₄ solutions is without effect. The effect is similar to that of methylation on phosphoric and carboxylic acid sites, and combination of the two procedures is not additive. Mast cell and cartilage staining are not significantly affected. Deoxyribonucleic acid is not extracted; the Feulgen reaction persists, though 1 hour hydrolysis is required vs 12–15 minutes on control formol material. Metal binding as revealed by staining in acetic hematoxylin remains demonstrable after 10 days heating in 2.5% K₂Cr₂O₇, but disappears in 6–16 hours in chromic, ferric and ferrous salt solutions. Chemical analysis reveals the continued presence of chromium at 4–5 % dry weight levels in liver and brain, alike after chromic acetate and potassium dichromate (24 hours, 60° C), though no histochemical Cr demonstration is obtained after Cr acetate. It is concluded that carboxyl and phosphoryl residues in tissue bind chromic (and iron) ions, which are then converted by heating to an unreactive insoluble form. Removal of demonstrable chromium with acid alcohol, sodium dithionite or ß-mercaptoethanol from dichromate treated tissues does not reverse the oxyphilia.Supported by National Cancer Institute Grant No. C4816, National Institutes of Health.     

Temperature acclimation in brook trout muscle: Adenine nucleotide concentrations, phosphorylation state and adenylate energy charge

Summary Cold acclimation in fish is associated with an elevation in metabolic rate. The present study investigates the role of adenine nucleotides and related compounds in metabolic regulation following temperature acclimation. Brook trout (Salvelinus fontinalis) were acclimated for 10 weeks to either +4°C or +24°C. Both groups of fish were exercised at 2.5 body lengths s^–1 for 2 weeks prior to sacrifice in order to control for differences in spontaneous activity.Concentrations of ATP, ADP, AMP, P _i and PC were approximately 2-fold higher in white than red muscles. Temperature acclimation had little effect on total adenine nucleotide concentration in either muscle type. In white fibres acclimation to 4°C results in a 39% increase in [ADP] and [AMP], a 35% decrease in [PC] (phosphorylcreatine), and no significant change in [P _i ]. In contrast temperature has little effect on concentrations of these compounds in red muscle.Parameters of metabolic control — adenylate energy charge ([ATP]+0.5 [ADP]/[ATP]+[ADP]+[AMP]), phosphorylation state ([ATP]/[ADP]·[P _i ]), and the ratios [ATP][ADP] and [ATP][AMP] — were significantly lower in cold- than warm-acclimated white muscle. The observed changes in phosphorylation state and [ATP][AMP] are consistent with an increase in mitochondrial respiration and glycolysis, respectively.In conclusion, changes in metabolites may be an important factor in producing an enhanced metabolic rate in cold-acclimated fish.     

Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state.     

Cardiac Sarcalumenin: Phosphorylation, Comparison with the Skeletal Muscle Sarcalumenin and Modulation of Ryanodine Receptor

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca²⁺ binding protein (HCP) with respect to: (i) Ca²⁺ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg²⁺ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La³⁺; (iv) phosphorylation is obtained with [γ-³²P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La³⁺. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca²⁺- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified. Received: 15 December 1998/Revised: 25 March 1999     

The effect of histochemical methylation on the phosphate groups of nucleic acids: Interpretation of the absence of nuclear basophilia

Summary The effect of hot methylation (hydrochloric acid-methanol) on nuclear stainability was investigated in order to determine whether the progressive loss of basophilia is due to methylation of the diester phosphate groups of nucleic acid.DNA spots on filter paper were unchanged in their stainability towards Toluidine Blue even after methylation for 4 days, while RNA, chondroitin sulphate and hyaluronic acid lost their affinity for this dye after 4 h methylation.In formalin-fixed sections, methylation for 4 h led to a loss of nuclear basophilia. There was no concomitant increase in nuclear relative to cytoplasmic stainability with Fast Green FCF at pH 9, as judged from the use of a comparison eyepiece, evaluation of colour transparencies or by microspectrophotometry. In contrast, extraction with trichloroacetic acid prior to or after methylation led to a much improved Fast Green staining of nuclei, comparable to the staining obtainable after treatment with trichloroacetic acid alone.In conclusion, there is no evidence that hot hydrochloric acid-methanol, as used in histochemistry, methylates the diester phosphate groups of nucleic acids. The loss of nuclear basophilia can be explained as a result of the excess positive charge on the chromatin following methylation of all the protein carboxyl groups. This effect is maximal after 3–4 h treatment with acid methanol at 60°C. Further methylation leads to depolymerization and extraction of DNA. RNA is depolymerized in less than 4h.