Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Acetylcholinesterase molecular forms in the fast or slow muscles of the chicken and the pigeon

Molecular forms of acetylcholinesterase (AChE) were examined in various skeletal muscles of the chicken and the pigeon. In chicken pectoralis m., AChE was found to be restricted to endplate containing segments, and no asymmetric form could be detected in aneural samples. In the chicken muscles studied, a relation has been established between globular (G1,G2,G4) forms or asymmetric (A8,A12) forms, and muscle fibre types. Asymmetric forms are preponderant in fast-twitch muscles, whereas in slow tonic muscles 80% of the AChE activity is due to globular forms. However, comparison with pigeon muscles shows that AChE chicken muscle patterns may not be generalized.     

Molecular Forms of Acetylcholinesterase: Regulation in a Testosterone-Sensitive Nerve-Muscle Axis

We measured the distribution of molecular forms of acetylcholinesterase (AChE) in muscles of a song bird, the zebra finch, and found a pattern similar to those reported in other vertebrates. As in other species, the most rapidly sedimenting form of the enzyme decreases to barely detectable levels following denervation. In the muscles of the syrinx, castration causes a large decrease in AChE activity, but has little or no effect on the relative abundance of AChE forms. This suggests that the number of AChE catalytic sites is changing without affecting the distribution of catalytic sites among the molecular forms. This is in marked contrast with the effect of denervation in the syrinx, which causes changes in the distribution of activity, as well as in total activity.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Germanium toxicity in selected bacterial and yeast strains

Summary The toxicity of germanium dioxide (GeO₂) to 21 bacterial and 13 yeast strains was investigated in liquid broth medium to obtain information on strains tolerant to high (1 to 2 mg/ml) GeO₂ concentrations.Arthrobacter sp. NRC 32005,enterobacter aerogenes NRC 2926,Klebsiella aerogenes NCTC 418 andPseudomonas putida NRC 5019 were tolerant to 1 mg/ml GeO₂.Bacillus sp. RC607 was able to grow in the presence of 2 mg/ml GeO₂ at pH 10 in broth culture. The yeastsCandida guilliermondii, Candida shehatae andPachysolen tannophilus were the most sensitive to GeO₂ as evidenced by their diminished growth rates at a GeO₂ concentration as low as 0.1 mg/ml. None of the yeast strains tested exhibited growth in the presence of 1 mg/ml GeO₂. The high pH of the medium containing germanium may be partially responsible for the growth inhibition of the yeast cultures. Select bacterial cultures previously exposed to 1 mg/ml GeO₂ could tolerate and grow better at 2 mg/ml GeO₂, suggesting the existence of very efficient adaptive mechanisms. The pH of the medium could modulate GeO₂ tolerance and this effect was found to be strain-dependent.     

五种水蛭对不同P^H值的生物效应

本文论述了使用常规试验方法研究5种水蛭:宽身舌蛭(Glossiphonia lata)、八目石蛭(Erpobdella octoculata)、光润金线蛭(Whitmania laevis)、尖细金线蛭(Whitmania acranulata)和日本医蛭(Hirudo nipponia)对12个pH值的24—96小时急性生物效应。结果表明:稻田3个种(尖细金线蛭,光润金线蛭和日本医蛭)均较湖泊近岸2个种(八目石蛭和宽身舌蛭)对pH值的变化要敏感,其中尖细金线蛭最敏感(pH6.0—7.2),八目石蛭的忍耐限度最宽(pH4.0—10.5);当pH值在9.5时,稻田3种水蛭的死亡率在40％以上,其中日本医蛭高达70％;当pH值在3.0时,湖泊近岸2种在24小时内全部死亡,日本医蛭仅死亡10％。日本医蛭是华中农村地区一种主要吸血蛭类,生石灰是一种从稻田消灭蛭类的有效手段,然而水的pH值必须不低于8.6。     

Comparison of muscle force,muscle endurance,and electromyogram activity during an expedition at high altitude

Handgrip force (HF), maximal pinch force (MF), muscle endurance (ME), and the median power frequency (MdPF) of the activity shown in the electromyogram (EMG) were studied at various altitudes in eight normal healthy subjects. MF and ME were measured between the index finger and thumb, and all measurements were obtained at altitudes ranging from 610 to 4860 m during an expedition in the Qinghai Plateau in China. With the change in altitude HF, ME, and MF showed no significant change. Compared to the MdPF at 2260 m on ascent, the MdPF at other altitudes showed a significant decrease (P<0.01). Thus, we conclude that muscle performance (HF, MF, and ME) was not affected by the environment at high altitude. However, MdPF was affected and the mean MdPF at 610 m after the expedition did not recover to initial values of MdPF. We suggest these results may have been affected by fatigue and chronic exposure to the hypobaric hypoxic environment, since the members of the expedition party expressed feelings of sluggishness and fatigue after the expedition.     

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996