Formation of Adventitious Buds in Decapitated Citrus Seedlings and the Effect of Some Growth Regulators

Young seedlings of various citrus species give rise, after decapitation,to adventitious buds at the cut end of the epicotyl. With suchtreatment, more buds formed on seedlings of sweet orange, sourorange, and grapefruit than of lemon, mandarin, and calamondin.Anatomical observations at the cut stem end show formation ofbuds occurring from tissues close to the cambium. Growth regulatorsapplied at the cut epicotyl end modified the patterns of budformation. 6-Benzylaminopurine (BAP) promoted bud formation;indole-3-butyric acid (IBA), abscisic acid (ABA), and gibberellicacid (GA₃) inhibited it. BAP reversed the inhibitory effectof GA₃. Gibberellic acid caused a typical elongation of thesprout axis. The use of decapitated seedlings as a tool forstudying stem differentiation in citrus is suggested.     

The Mechanism of Abscisic Acid-induced Proline Accumulation in Barley Leaves

When leaf blades of fully expanded second leaves of barley (cv. Prior) were excised and incubated with the cut end in a 20 milligram per liter solution of abscisic acid, they accumulated proline at the rate of about 1 micromole per hour per gram fresh weight after a 3- to 4-hour lag. This accumulation occurred reproducibly only if leaves were pretreated by placing the cut end in a solution consisting of 50 millimolar sucrose and 1 millimolar glutamate. Treated leaves were taken from plants which had been in the light for 24 hours.     

The senescence retarding ability and metabolism of trihydroxy-zeatin in Avena leaf segments

6-(2,3,4-Trihydroxy-3-methylbutylamino) purine, trivial name trihydroxy-zeatin (THZ), an oxidation product of zeatin was applied via the epidermis or cut end of Avena leaf segments. THZ did not retard senescence of the segments in either case. With epidermal application THZ remained unmetabolised while the cut end method of application resulted in its metabolism to more polar THZ metabolites of unknown identity. The Avena leaf tissue did not apparently cleave the isoprenoid side chain of the THZ molecule.     

Asymmetric behavior of severed microtubule ends after ultraviolet- microbeam irradiation of individual microtubules in vitro

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.     

Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy

Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 microm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.     

Ionic dependence of the axotomy-induced long-lasting firing in an identified crayfish motoneuron

I have reported previously that axotomy of an identifiable anal motoneuron of crayfish Procambarus clarkii induces a long-lasting firing and that a prolonged depolarizing pulse to its cut end can induce a similar response. In this study, I confirmed that this stimulus is comparable to axotomy; the frequency of stimulus-induced firing increases linearly with the stimulus intensity and its firing pattern is the same as that following axotomy. Then, when the cut end was bathed for more than 1 hr in test solutions, it was examined whether the stimulus to the cut end induces or blocks the response. Na(+)-free saline (Tris(+) replaced Na(+)) or TTX (3 x 10(-7) M) reversibly blocked the response within 30 min. By contrast, Mn(2+) saline (40 mM Mn(2+) replaced Ca(2+)) or Ca(2+)-free salines (Mg(2+) or 1 mM EDTA replaced Ca(2+)) cannot block the response, but instead increased the firing frequency. These results obtained with stimulus were confirmed also by those with axotomy. I concluded that axotomy-induced firing, which occurs locally at its cut region, is primarily responsible for voltage-dependent Na(+) conductances, but not for Ca(2+) ones.     

Application of leaf extract causes repetitive action potentials inBiophytum sensitivum

A wound stimulus evoked a number of repetivite action potentials in the leaf ofBiophytum sensitivum. When the cut end of a leaf was immersed in a leaf extract, the resulting repetitive action potentials continue for a long time. These repetitive action potentials disappeared immediately when the leaf extract, which contains a proposed stimulant, was removed and the cut end was washed with water.     

Residual strains in porcine and canine trachea

Residual strains exist in canine and porcine tracheas. They are revealed by cutting the trachea first perpendicular to its axis into rings, then radially into sectors. Each sector is characterized by an opening angle which is defined as the angle subtended between two radii joining the middle point of the inner wall to the tips of the inner wall. The trachea being non-axisymmetric, the opening angle depends on the position of the radial cut. The trachea being also nonuniform in the axial direction, the opening angle varies along the length of the trachea. In the dog, the opening angle of the trachea cut at the anterior position (cartilaginous) is about 100 degrees at the larynx; it increases fairly linearly to 180 degrees midway down the trachea; then increases slowly to about 200 degrees at the lower end where the trachea bifurcates into the main bronchi. Dog trachea cut in the posterior (muscular) position have an opening angle of about 50 degrees at the larynx, which increases to about 70 degrees three-quarters of the way down the trachea, then drops to 60 degrees at the lower end. In the pig, the opening angle of the trachea is much smaller, the values at anterior and posterior cuts are similar (without significant difference), and their mean value decreases from about 15 degrees at the laryngeal end to about 5 degrees at the lower end. These species and regional differences are discussed in relation to tracheal geometry and structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of air embolism in xylem conduits of pre-defined diameter.

A new method is presented that enables the induction of embolisms in a fraction of all xylem vessels, based on diameter, at one cut end of a stem segment. The method is based on the different capillary characteristic of xylem vessels of different cross-sectional size. To verify the method, air embolisms were induced in cut xylem vessels of chrysanthemum (Dendranthemaxgrandiflorum Tzvelev cv. Cassa) stem segments at different xylem tensions and compared with the distribution of gas-filled vessels as visualized by cryo-scanning electron microscopy (Cryo-SEM). At -6 kPa xylem pressure, air-entrance was only induced in large diameter vessels (>30 microm), while at -24 kPa embolisms were induced in almost all xylem vessels (>10 microm). Although the principle of the embolization method worked well, smaller diameter vessels were observed to be embolized than was expected according to the calculations. The role of cross-sectional shape and contact angle between xylem sap and vessel wall at the menisci are discussed. After correction for the observed (diameter independent) deviation from circularity of the cross-sectional vessel shape the contact angle was calculated to be approximately 55 degrees. Hydraulic resistance (Rh) measurements before and after embolization showed that the effect of embolizing only large diameter cut xylem vessels had only a small influence on overall Rh of a stem segment. Embolizing all cut xylem vessels at one cut end almost trebled overall Rh. The difference was discussed in the light of the networking capacity of the xylem system.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Direction of Travel of RecBc Recombinase through Bacteriophage Lambda DNA

We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction. The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange. In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end.     

Cell-to-cell tracer movement in cardiac muscle

Summary An attempt was made to label injured cardiac muscle cells by exposing them to two electron-opaque tracers, ruthenium red and lanthanum nitrate. To do this, false tendons of sheep hearts containing strands of Purkinje fibers were sectioned, allowed to heal, and then exposed to the tracer during fixation. After this treatment, a group of cells near the cut end were found to be labelled intracellularly with the tracers while the remaining cells in the strand were unlabelled.For comparison, several false tendons were fixed briefly in glutaraldehyde before being cut and then exposed to the tracer. With lanthanum, the results were similar to those obtained when the cells had been damaged prior to fixation. However, when ruthenium red was used as the tracer, it penetrated much further into the cellular strand, its intensity gradually diminishing with distance from the cut end. This finding of apparent dye-coupling in fixed tissue was surprising since it has been suggested that glutaraldehyde fixation converts all communicating junctions to the uncoupled state.Dye-coupling of fixed tissue with ruthenium red as a tracer was seen also in frog atrial trabeculae.Gap junctions between injured (and presumably uncoupled) sheep heart Purkinje cells were compared to gap junctions between uninjured control cells in thin sections. No difference was detected.     

The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation.

Rates of protein synthesis were significantly lower in the cut soleus and extensor digitorum longus muscles than in their uncut counterparts. Rates of protein degradation were significantly higher in cut soleus muscles, but not in cut extensor digitorum longus muscles as compared with their uncut controls. Concentrations of ATP and phosphocreatine were significantly lower in cut soleus and extensor digitorum longus muscles after incubation in vitro in contrast with respective control uncut muscles. These data indicate that cutting of muscle fibres alters rates of protein synthesis and degradation, in addition to altering concentrations of high-energy phosphates. Since these findings stressed the importance of using intact muscles to study protein metabolism, additional studies were made on intact muscles in vitro. Stretched soleus muscles had higher concentrations of high-energy phosphates at the end of an incubation period than did unstretched muscles. However, the length of the soleus, extensor digitorum longus and diaphragm muscles during incubation did not affect rates of protein degradation.U     

Rhizoid differentiation in Spirogyra: position sensing by terminal cells

Some species of Spirogyra anchor themselves to the substrate by differentiating rhizoids. A rhizoid is differentiated only from the terminal cell, suggesting that this cell can recognize its terminal position in a filament. In the present study, we have analyzed the mechanism for position sensing by the terminal cell. When a filament is cut, a new cell occupies the terminal position, and three phenomena are induced: (1) the cell wall of the cut cell detaches from the new terminal cell; (2) adhesive material is secreted by the terminal cell; and (3) the terminal cell begins to differentiate a rhizoid via tip growth. All of these phenomena were inhibited by adding sorbitol to the external medium, suggesting that turgor pressure is involved in position sensing by the terminal cell. The inhibition by sorbitol was reversible. Upon cutting a filament, the distal end of a new terminal cell became convex. However, when a filament was cut in the presence of sorbitol, the distal end of a new terminal cell became less convex. Either treatment with Gd(3+) or decrease in extracellular Ca(2+) resulted in inhibition of all these phenomena, suggesting possible involvement of stretch-activated ion channel in position sensing by terminal cells.     

Acetylcholinesterase activity in the nervous system of normal planaria and during regeneration

The acetylcholine esterase (AChE) activity was studied in the nervous system of planarians in the normal state and during regeneration. During the regeneration of the cephalic body end, the AChE appears in the cells of the newly formed ganglion on the 5th day. During the regeneration of caudal body end, the AChE activity in the residual ganglion after the cut exceeds the normal level. The possible role of the ACh system in the processes of planarian regeneration is discussed.     

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

The effect of tobacco tax cuts on cigarette smoking in Canada

OBJECTIVE: To assess the effect of the tobacco tax cuts made in 1994 on the smoking habits of Canadians. DESIGN: Population-based retrospective cohort study. DATA: Data from the Survey on Smoking in Canada conducted by Statistics Canada on 11,119 respondents 15 years of age and older, who were interviewed about their smoking habits on 4 occasions, approximately every 3 months from January 1994 to February 1995. OUTCOME MEASURES: Changes in smoking prevalence, incidence, quit rates and mean number of cigarettes smoked per day in the provinces where tobacco taxes were cut and in those where taxes were not cut. RESULTS: During the survey, smoking prevalence decreased in all provinces, whether or not cigarette taxes had been cut. However, the prevalence of smoking was greater in the provinces where tobacco taxes had been cut than in those where they had not, and this difference increased from 2.0% at the beginning of the survey to 3.4% by the end (p < 0.001). In addition, rates of starting cigarette smoking were higher and smoking quit rates were lower in the provinces where taxes had been cut than in those where taxes had not been cut. CONCLUSION: Although smoking rates are declining in Canada, tobacco tax cuts appear to have slowed the rate of decline by inducing more nonsmokers to take up smoking and leading fewer smokers to quit.     

Formation and transport of 1-aminocyclopropane-1-carboxylic acid in pea plants

The formation and transport of free 1-aminocyclopropane-1-carboxylic acid (ACC) and conjugated ACC [1-(malonylamino)cyclopropane-1-carboxylic acid; M-ACC] was studied in pea plants. Excision and dark incubation induced ACC and M-ACC synthesis in stem segments, including the second node. At similar rates as in segments, ACC and M-ACC were formed near the cut surface in stems after decapitation, leading to a transient increase in both compounds in the node adjacent to the cut. The maximum level of M-ACC at 6 hr exceeded that of ACC at 3 hr. Seven days after decapitation, total M-ACC in the shoot returned to the level in the control plants. Over the same period of time, M-ACC accumulated in the roots in amounts comparable to those previously observed in the shoot. It is concluded that M-ACC formed near the cut is transported basipetally, and that the roots act as a sink. Both the increase in ACC and M-ACC in the node after decapitation and the degree to which growth of lateral shoots was inhibited by ACC applied to the cut end increased with advancing age of the plant.     

The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern.

In cotyledon cultures of Brassica juncea, shoots and roots invariable differentiate at the cut end of the petiole. Organogenesis occurred only if the proximal cut end of the petiole was in contact with the medium. In the absence of the petiole, differentiation from the lamina was rare. Hence investigations were carried out to study the influence of the cotyledonary lamina on regeneration of shoots and roots from the petiolar cut end. The lamina tissue was surgically removed from the cotyledon explants at different durations (0–10 days) after culturing them on either root-forming (basal medium) or shoot-forming (basal medium containing 5.0 M N⁶-benzyladenine) media. The differentiation of roots or shoots from the petioles was dependent on the presence of the lamina for at least 7 days of culture. Quantitative removal of the laminar tissue had a corresponding negative effect on shoot bud differentiation from the petiole. The nature of the lamina factor was found to be auxin-like.