The Effect of Recombinant Neurotoxins from the Sea Anemone <Emphasis Type="Italic">Anthopleura</Emphasis> sp. on Sodium Currents of Rat Cerebral Cortical Neurons

We have investigated the action of the recombinant neurotoxins, named Hk7a and Hk2a, whose amino acid sequences differ only in two positions, isolated from the sea anemone Anthopleura sp., on neuronal sodium currents using the whole-cell voltage-clamp techniques. The rat cerebral cortical neurons in primary culture were used for this study. In our experiments, these cells all express tetrodotoxin-sensitive (TTX-S) sodium currents. Under the voltage-clamp condition, application of Hk7a and Hk2a reduced the sodium channel current amplitude and shifted the voltage dependence of activation to more positive potential; while Hk7a produced no significant effect on the voltage at which 50% of the channels were inactivated, Hk2a caused profound hyperpolarizing shift of the voltage-dependent inactivation. Also, both Hk7a and Hk2a increased the time course of recovery from inactivation. In kinetic studies, whereas application of Hk2a slows the time to peak of voltage-gated sodium channel, the time course of fast and slow inactivating component, no significant effect was observed in Hk7a. These results suggested that the difference of key amino acid between Hk7a and Hk2a might contribute to their different action; therefore, they could be used as pharmacological tool to study the structure and function of voltage-gated sodium channel. Hui Xiang, Wucheng Tao, Lei Wang, and Fang Wang have contributed equally to this work.     

The microcirculatory bed of the normal rat thyroid and during hypokinesia based on scanning electron microscopy data on corrosion preparations

In 12 mature white rats with body mass of 180-230 g, that are kept in tight pencil-cases for 10, 30 and 90 days (3 rats make the control), by means of scanning electron microscopy of corrosive preparations reorganizations of three-dimensional spatial organization of the thyroid microcirculatory bed are studied. In intact animals single-layered network of the perifollicular capillaries consists of widely anastomotic blood microvessels and makes 68 +/- 7% of the follicular surface. At hypokinesia (Hk) lasting for 10 days, the perifollicular capillaries are sharply dilated, the capillary network area makes 74 +/- 4%. A large amount of processes appear on the capillary walls. On the 30th day of Hk unequal distribution of capillaries on the follicular surface is noted, that is heterogeneity in organization of the perifollicular capillary networks is manifested. In 90 days of Hk reduction of the capillaries is recorded, rarefied pericapillary network prevails, twistedness of the capillaries is clearly manifested, their complex branching decreases. The capillary network area makes 54 +/- 3% of the follicular area. A large amount of pin-shaped protrusions of the capillary wall appear.     

棉纤维发育过程中细胞壁超微结构变化的研究

应用X射线衍射法研究了棉纤维发育过程中细胞壁超微结构变化的动态规律.其结果是:次生壁S_2层的平均微纤丝螺旋角随花后生长天数的增加而逐渐变小:纤维素微晶粒间的取向度逐渐变大;花后5—14天内,结晶度缓慢增加,14—17天内陡然增加,17天后缓慢趋向最大值.     

Experimental regeneration of the lungs

In experiments on 150 guinea pigs, processes of lung wound healing were followed and the more active cellular sources of regeneration were identified. Measurements of cyclic adenosine monophosphate components in the course of lung healing indicated that histologic and electron-microscopic studies would be more informative if done at days 1, 3, 4, 7, and 14 after lung damage. Both cellular and intracellular mechanisms of regeneration were involved in the healing processes. The type II pneumonocyte be designated as a cambial cell of pulmonary parenchyma, for this cell displays the highest proliferative potency and is responsible for the epithelial lining of newly formed alveoli. The formation of these is shown to be assured through functional activity of all cellular alveolar elements (involving intracellular hyperplasia and hypertrophy of ultrastructures and especially dynamic morphological and functional alterations in osmiophilic bodies of type II pneumonocytes) and capillary neoformation.     

Genetic Manipulation of an Abnormal Jump Response in Drosophila

The hyperkinetic mutants, Hk(1) and Hk(2), jump and fall over when an object moves near them. This behavior, the kinetogenic response, has been measured by the experimenter moving his hand above a vial containing a single fly and scoring the number of positive responses in fifty trials. The response is higher in Hk(1) than in Hk(2) and has remained so over a period of several years and in different genetic backgrounds. The Hk(1)/Hk(2) heterozygote also responds to movements, establishing the allelism of the two mutants.-When Sh(5), a shaker mutant at another locus on the X chromosome, is introduced into the same chromosome as Hk(1) or Hk(2), the response is reduced in proportion to the number of Sh(5) mutant genes added.-When Hk(1) or Hk(2) is heterozygous with a deficiency for the hyperkinetic region of the X chromosome, the expression is more abnormal than the respective Hk(1) or Hk(2) homozygote. This shows that the mutant genes are producing an altered gene product, or less of the normal, since one mutant gene by itself has a more abnormal expression than two. A tentative explanation has been offered for the observed mutant behaviors.     

Parallel-fibered muscles transplanted with neurovascular repair into bipennate muscle sites in rabbits.

Experiments were performed on 20 New Zealand White male rabbits. Our hypotheses were that (1) latissimus dorsi (LTD) muscles transplanted into the site of a bipennate rectus femoris (RFM) muscle with neurovascular repair would retain their parallel-fibered structure and (2) the parallel-fibered structure of latissimus dorsi grafts would reduce their total fiber cross-sectional area and adversely affect force development relative to that of bipennate rectus femoris grafts and muscles. Compared with their respective donor muscles, 120 to 150 days after grafting, latissimus dorsi and rectus femoris grafts showed no change in the number of fibers and a decrease in the mean single-fiber cross-sectional area to approximately 70 percent. The latissimus dorsi grafts, which remained parallel-fibered, developed maximum forces 34 and 23 percent of the values for fully activated rectus femoris grafts and muscles, respectively. The deficit in the maximum force of the latissimus dorsi grafts resulted primarily from the smaller total-fiber cross-sectional area as a result of the parallel-fibered structure.     

Cellular mechanisms of compensatory processes of the kidney in relation to the mass resected

As a result of comparative investigation of compensatory processes in the kidneys at various degree of their resection, the most general regularities in these processes have been revealed: an initial functional strain of renal cells and their ultrastructures, the secondary increase in hypertrophy and hyperplasia of the renal cells and their organelles. The degree of manifestation of these regular reactions directly depends on the mass of the resected part of the kidney. Subtotal resection of the kidney (80%) is an operation that gets out of the limits of the organ's compensatory possibilities. A sharp decrease, up to complete disappearance of its cellular division in 2-3 weeks after the operation is of great importance. Mechanism for development of renal insufficiency is evidently connected with absence of cellular renewal against the background of their hyperfunction and their quick worn out, that is, evidently, resulted in deficiency of renal epithelium and functional defectiveness connected with it in the remained renal mass.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

The thoracic mechanism for eclosion and digging during the extrication behaviour of Diptera

ABSTRACT. Dipteran flies escape from the puparium by coordinated contractions of all segments of the body. Whereas special eclosion muscles have been identified in the abdomen and head, none has previously been described in the thorax. Three pairs of large thoracic muscles are described in this paper which are involved solely with eclosion. They have ultrastructures consistent with an ability to supercontract and all three degenerate within 48 h following escape from the puparium. Recordings of electrical activity show them to be rythmically active, coincident with thoracic contractions during eclosion. Many of the non-fibrillar flight muscles are also incorporated in the eclosion motor pattern and have a precise sequence of activity. Following escape there is a rapid switch from eclosion to flight motor and this is discussed with reference to afferent mediation and changing inputs to the muscles.     

Effects of recombinant neurotoxins on single Na+ channels in isolated rat hippocampal neurons

Four recombinant neurotoxins Hk2a, Hk7a, Hk8a, Hk16a, originally from a sea anemone species Anthopleura sp., were obtained by fusion expression of their genes in Escherichia coli. These neurotoxins were composed of 47 amino acid residues, among which the differences were found at positions 14, 22, 25, and 37, respectively. The effects of the four neurotoxins on single‐channel current of sodium in rat hippocampal neurons were studied by cell‐attached patch clamp. Each neurotoxin 2 μM could modulate the sodium channel by prolonging the opening dwell time and increasing the open probability, but did not change the amplitude of sodium channel currents. Based on the studies of the structure–function relationship, we found that Hk7a displayed the biggest increase of the open probability because His14 (from Arg14) makes its structure seem more compact in comparison with the other three toxins and Ap‐A. Phe25 (Hk8a, Hk16a), which varied from Ala25 (Hk2a, Hk7a), showed that phenyl group might interfere with other key amino acid residue to decrease the activity of toxins. Arg37 (from His37) in Hk8a contributed to decrease of open probability. In our work, it was shown that these important amino acid sites might provide a reliable proof for the future pharmaceutical design. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:244–255, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20285     

An ultrastructural and physiological investigation of the retractor muscles of Gené's organ in the cattle ticksBoophilus microplus andAmblyomma variegatum

The ultrastructures of the retractor muscles of Gené's organs in the cattle ticksBoophilus microplus andAmblyomma variegatum are described. The innervation, neuromuscular junctions, and insertions of the muscles are also described. The retractor muscles are important in controlling the actions of Gené's organ, the egg waxing organ in ticks, during oviposition in the female. The ultrastructural features of the muscles are typical of arthropod muscles, and the nerve terminals at the neuromuscular junctions contain small electron lucent synaptic vesicles with a diameter of 50 nm, and also larger dense core vesicles with a diameter of 100 nm. Evidence is presented implicatingl-glutamate andl-aspartate as putative excitatory transmitters at the tick neuromuscular junction. The excitatory post-synaptic potentials recorded in the muscles were abolished in the presence of low concentrations ofl-glutamic acid andl-aspartic acid, but were unaffected by acetyl-choline, 4-aminobutyric acid and octopamine, suggesting that glutamic acid and aspartic acid interact with receptors on the muscle membrane.     

Features of the development of the pituitary-thyroid system of the Wrangel Island hoofed lemming Dicrostonyx torquatus at stages of growth and peak population

By means of morphometric techniques, peculiarities in formation of the hypophyseal-thyroid system during prenatal and early postnatal periods have been studied in lemmings (Dicrostonyx torquatus) from the Wrangel island at growth stages and at the peak of the population quantity. Folliculoformation rate, time when the organ reaches the definitive structure, periods for establishing connection with the regulating center--the anterior hypophyseal lobe, correlation of differentiation rates of histo- and ultrastructures of the organ distinctly react to changes in the density population of the animals. Periods of initiation of folliculoformation are early and comparable at both stages of the population cycles. However, further at the peak of the population quantity, an essential retardation in the development of both histo- and ultrastructures of the thyroid cells is observed; formation of the histostructure significantly overtakes differentiation of the ultrastructures composing its cellular elements. The follicular structure begins to form only in newborn animals, the folliculogenesis completes on the 1st-6th days of the postnatal period. At the stage of growth of the population quantity, the folliculogenesis lasts 2 days (the 15th-17th days of the prenatal development), the organ's structure becomes definitive on the 17th day, at the same period connections are established with the anterior hypophyseal lobe. The role of the thyroid hormones in formation of reprodactive and adaptive potencies of the organism at various stages of the population cycle is discussed.     

Flowering of Stylosanthes guianensis in Relation to Juvenility and the Long-Short Day Requirement

Seedlings of Stylosanthes guianensis var. guianensis were grownin long (14 h) days in five temperature regimes for varyingperiods before transfer to short (11 h) days at 30 ?C/21 ?C.The juvenile phase before seedlings responded to inductive conditionswas c. 45–50 d, 50–60 d and 60–70 d for cv.Schofield, cv. Cook and C.P.I. 34906 respectively, which ispositively related to their critical photoperiod for flowering.Temperatures favourable for growth (e.g. 30 ?C/26 ?C) reducedthe juvenile phase in C.P.I. 34906 and in Cook, which did notflower in 11 h days unless previously exposed to more than 18long days. In a second experiment cv. Cook was confirmed as a long-shortday plant. Seedlings were grown for 50 d in a glasshouse withnatural daylength extended to 13, 14, 16 or 24 h before transferto 12 h photoperiods. Cook floral development was positivelyrelated to daylength provenance before transfer and plants incontinuous 12 h did not flower. Shortening daylength after 48 cycles of 12 h to 11.75 h didnot result in continued floral development in Cook plants butcv. Graham plants were initiated or transitional by 75 d. Key words: Stylosanthes guianensis, Photoperiod, Temperature, Flowering     

The Development of Lateral Root Primordia in Vicia faba L. and Their Response to Colchicine

Lateral root primordia in i are first initiated 2–3 daysfollowing the onset of germination, after which they take 5.17–6.35days to complete their development and emerge as lateral roots.Variation in the amount of time elapsing between primordiuminitiation and emergence as a lateral is probably a reflectionof the cell number attained by any one primordium at the timeof emergence. The number of primordia produced per cm of primaryroot growth (5.35–6.65) was not affected by variationin the rate of root elongation, although the number of primordiaproduced each day increased with increase in the rate of rootgrowth. In colchicine-treated roots, the amount of time between primordiuminitiation in the C-tumour and the subsequent emergence of alateral (5.43–6.43 days) was similar to the value obtainedin control roots. Primordia which were present at the time ofcolchicine treatment responded to treatment in a number of differentways, depending on the stage of development reached. Primordiain the first 2.66 days of their development die following treatment;those between 2.66 and 3.69 days old have their developmentinhibited but stay alive; primordia which have been developingfor 3.69–4.91 days following initiation grow out as straightlaterals, while those between 4.91 and 5.77 days old form C-tumoursand emerge as inhibited laterals.     

The COX-2 pathway is essential during early stages of skeletal muscle regeneration

Skeletal muscle regeneration comprises several overlapping cellular processes, including inflammation and myogenesis. Prostaglandins (PGs) may regulate muscle regeneration, because they modulate inflammation and are involved in various stages of myogenesis in vitro. PG synthesis is catalyzed by different isoforms of cyclooxygenase (COX), which are inhibited by nonsteroidal anti-inflammatory drugs. Although experiments employing nonsteroidal anti-inflammatory drugs have implicated PGs in tissue repair, how PGs regulate muscle regeneration remains unclear, and the potentially distinct roles of different COX isoforms have not been investigated. To address these questions, a localized freeze injury was induced in the tibialis anterior muscles of mice chronically treated with either a COX-1- or COX-2-selective inhibitor (SC-560 and SC-236, respectively), starting before injury. The size of regenerating myofibers was analyzed at time points up to 5 wk after injury and found to be decreased by SC-236 and in COX-2^–/– muscles, but unaffected by SC-560. In contrast, SC-236 had no effect on myofiber growth when administered starting 7 days after injury. The attenuation of myofiber growth by SC-236 treatment and in COX-2^–/– muscles is associated with decreases in the number of myoblasts and intramuscular inflammatory cells at early times after injury. Together, these data suggest that COX-2-dependent PG synthesis is required during early stages of muscle regeneration and thus raise caution about the use of COX-2-selective inhibitors in patients with muscle injury or disease. prostaglandins; nonsteroidal anti-inflammatory drugs; muscle growth; inflammation; satellite cells     

Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Effect of ecdysterone and juvenoid on the developmental involution of flight muscles in Acheta domestica

Morphogenesis and degeneration of the flight muscles in Acheta domestica was studied. The dorso-longitudinal flight muscles (DLMs) degenerate during the fourth day after adult ecdysis and the dorso-ventral flight muscles (DVMs) on the fifteenth day. In the presence of an intact innervation the degeneration of the DLMs can be retarded for 2 days by the injection of ecdysterone into very young adults. This retardation may also result in hypertrophy of the muscle fibres. The injection of ecdysterone, even in high doses, did not affect the flight muscle remnants. No notable changes have been found in the degeneration of DLMs by ovarectomy. Thus, the degeneration of flight muscles and the development of ovaries appear to be independent processes.The DLMs are homogeneous in fibre pattern in respect to succinic dehydrogenase, an important oxidative enzyme, and to ATPase activity, but the muscle fibres do not show any phosphorylase activity.