Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

Biochemical correlates of fatigue. A brief review

Muscle fatigue, defined as a decreased force generating capacity, develops gradually during exercise and is distinct from exhaustion, which occurs when the required force or exercise intensity can no longer be maintained. We have reviewed several biochemical and ionic changes reported to occur in exercising muscle, and analysed the possible effects these changes may have on the electrical and contractile properties of the muscle. There is no evidence that substrate depletion can account for the decreased force generating capacity, but this factor may be important for the rate of energy turnover and be a major determinant for endurance. Increased concentration of inorganic phosphate and hydrogen ions will depress the force generating capacity, but since fatigue can develop gradually without accumulation of these ions they can only be important when aerobic ATP production is insufficient to support the contractions. Evidence is presented showing that a disturbed balance of K+ alone might cause depolarisation block at high stimulation frequencies, but extracellular K+ accumulation does not increase gradually during prolonged dynamic or static exercise, and is therefore not closely related to fatigue. The repeated release of Ca2+ from the sarcoplasmic reticulum (SR) during muscular activity is suggested of Ca2+ by the mitochondria, increasing with stimulation frequency and duration and possibly also deteriorating mitochondrial function. We therefore speculate that decreased Ca2+ availability for release from SR might contribute to a gradual decline in force generating capacity during all types of exercise.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Age- and sex-related differences in lipid peroxidation of mouse cardiac and skeletal muscles

1. The purpose of the present study was to characterize age- and sex-related changes in lipid peroxidation capacities and enzymatic antioxidants of cardiac and skeletal muscles in NMRI-mice (Mus musculus). 2. Lipid peroxidation rates (unstimulated and enzymatic/iron-stimulated) strongly decreased in skeletal muscle during ageing. 3. Unstimulated lipid peroxidation rate but not that of stimulated, also decreased in cardiac muscle. 4. The total level of Fe2+/ascorbate-stimulated non-enzymatic lipid peroxidation was not, however, affected by ageing. 5. The activity of catalase slightly increased in cardiac muscle and that of glutathione peroxidase in skeletal muscle during ageing. 6. Unstimulated lipid peroxidation rate was significantly higher in the skeletal muscle of male than female mice. 7. Correspondingly, the Fe2+/ascorbate-stimulated lipid peroxidation capacities of microsomal and mitochondrial fractions of skeletal muscle were significantly higher in male mice. 8. The activity of glutathione peroxidase as well as the concentration of lipofuscin were higher in the cardiac muscles of female than male mice.     

Splicing and spliceosome formation of the yeast MATa1 transcript require a minimum distance from the 5'' splice site to the internal branch acceptor site.

Small deletions of 6, 7, and 12 nucleotides introduced between the 5' splice site and the internal branch acceptor site of the first intron of the yeast MATa1 gene completely abolish accurate splicing in vitro in these constructs. Splicing only occurs at an alternative 5' splice site which was found in the first exon of the MATa1 gene and which is used both in vivo and in vitro. The splicing defect cannot be cured by expanding the distance from the branch point to the 3' splice site. If the alternative 5' splice site is deleted as well in these constructs, neither spliced products nor spliceosomes are formed. Our findings especially lead to the conclusion that a minimum distance between the 5' splice site and the internal branch acceptor site of the intron is required for the formation of splicing complexes and for accurate splicing.     

The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin_β and profilin-actin_γ. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg²⁺, and can be crystallized. From the isolated profilin-actin complexes the β- and γ-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes.

We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.