Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

Summary An amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.     

Multichannel transmission of proprioceptive input to motoneurons

A model has been developed to simulate the parallel channels of muscle spindles and their monosynaptic connections to a homonymous motoneuron in the turtle. Input to the model is muscle length and stimulation, output is motoneuronal membrane potential. Quality of transmission is greatly dependent upon dispersive properties of the system. The contributions of different dispersive features are compared and also cumulative effects are considered. Reference is made to conditions which are found in actual movements.     

In vivo dopamine receptor binding studies with a non-radioactively labeled agonist,dipropyl-5, 6-ADTN

Evidence is presented that a low dose of peripherally administered N, N-dipropylamino-5, 6-dihydroxytetralin (DiPr-5, 6-ADTN) specifically labels dopamine (DA) receptors in rat brain.Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60–70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, α-adrenergic or β-adrenergic antagonists. DiPr-5, 6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Loss of interleukin-10 exacerbates early Type-1 diabetes-induced bone loss

Type-1 diabetes (T1D) increases systemic inflammation, bone loss, and risk for bone fractures. Levels of the anti-inflammatory cytokine interleukin-10 (IL-10) are decreased in T1D, however their role in T1D-induced osteoporosis is unknown. To address this, diabetes was induced in male IL-10 knockout (KO) and wild-type (WT) mice. Analyses of femur and vertebral trabecular bone volume fraction identified bone loss in T1D-WT mice at 4 and 12 weeks, which in T1D-IL-10-KO mice was further reduced at 4 weeks but not 12 weeks. IL-10 deficiency also increased the negative effects of T1D on cortical bone. Osteoblast marker osterix was decreased, while osteoclast markers were unchanged, suggesting that IL-10 promotes anabolic processes. MC3T3-E1 osteoblasts cultured under high glucose conditions displayed a decrease in osterix which was prevented by addition of IL-10. Taken together, our results suggest that IL-10 is important for promoting osteoblast maturation and reducing bone loss during early stages of T1D.     

Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast

Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Steady-state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo-COX in the SHY1-disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D-PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Deltashy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D-PAGE.     

Presenting symptoms and clinical features in 130 patients with the velo-cardio-facial syndrome. The Leuven experience

During the last 5 years, we diagnosed in Leuven 130 patients with a 22q11 deletion. The deletion was familial in 14 out of 110 index patients (12%), which is significantly less compared to previous studies. In 10 patients, the deletion was maternal, in 4 patients paternal. A cardiac defect was the main presenting symptom in 49% of patients. The other patients were ascertained through developmental delay (16%), behavioural disturbances (7%), otorhinolaryngological manifestations (6%), psychiatric manifestations (3%) and mental retardation (2%). In one patient hypocalcemia was the presenting symptom. In another patient the severe immune deficiency led to diagnosis. Most patients presented a wide variety of the classical features of the Velo-Cardio-Facial syndrome. Velopharyngeal incompetence, learning difficulties or mostly mild mental retardation were almost always present, whereas clinical significant hypocalcemia or immune disturbances were rare. Previously un(der)recognised features include polyhydramnios, renal malformations and laryngotracheamalacia or laryngeal stenosis.     

Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes

Myoglobin plays various roles in oxygen supply to muscle mitochondria. It is difficult, and in some cases impossible, to study the relationship between the myoglobin concentration and the oxidative capacity of individual muscle cells because myoglobin has to be fixed in situ whereas determination of oxidative capacity, for example, succinate dehydrogenase activity, requires unfixed cryostat sections. We have investigated whether a vapour-fixation technique allows the use of serial sections to study the relationship between myoglobin and succinate dehydrogenase activity. The technique is used to study a rat soleus muscle, two human skeletal muscle biopsies and biopsies of two patients with chronic heart failure, and in a control and hypertrophied rat heart. Staining intensities were quantified by microdensitometry. The absorbance values were calibrated using sections cut from gelatine blocks containing known amounts of myoglobin. The results show that it is possible to use serial sections for the determination of the myoglobin concentration and succinate dehydrogenase activity, and indicate that myoglobin can lead to a substantial reduction (18–60%) of the extracellular oxygen tension required to prevent an anoxic core in muscle cells.     

Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.