Tail regeneration and ependymal outgrowth in the adult newt, Notophthalmus viridescens, are adversely affected by experimentally produced ischemia

Spinal axons of the adult newt will regenerate when the spinal cord is severed or when the tail is amputated. Ischemia and associated hypoxia have been correlated with poor central nervous system regeneration in mammals. To test the effects of ischemia on newt spinal cord regeneration, the spinal cord and major blood vessels of the newt tail were severed 2 cm caudal to the cloaca as a primary injury. This primary injury severely reduced circulation in the caudal direction for 7 days; by day 8, circulation was largely restored. After various periods of time after primary injury, tails were amputated 1 cm caudal to the primary injury (in the area of ischemia) and tested for regeneration. If the tail was amputated within 5 days of the primary injury, regeneration did not occur. If amputation was 7 days or longer after the primary injury, a regenerative response occurred. Histology showed that in the non-regenerating tails the spinal cord and associated ependyma, known to be important to tail regeneration, had degenerated in the rostral direction. Such degeneration was prevented when tails were first amputated and allowed to form blastemas before the primary injury. The data indicate that the first 5-7 days of blastema formation are particularly sensitive to compromised blood flow (ischemia/hypoxia). It follows that mechanisms must be present in the adult newt to reduce ischemia to a minimum and thus allow ependymal outgrowth and tail regeneration.     

How Listeria exploits host cell actin to form its own cytoskeleton. I. Formation of a tail and how that tail might be involved in movement

After Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria then nucleates actin filaments from its surface. These actin filaments rearrange to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. Since individual actin filaments appear to remain in their same positions in the tail in vitro after extraction with detergent, the component filaments must be cross-bridged together. From careful examination of the distribution of actin filaments attached to the surface of Listeria and in the tail, and the fact that during and immediately after division filaments are not nucleated from the new wall formed during septation, we show how a cloud of actin filaments becomes rearranged into a tail simply by the mechanics of growth. From lineage studies we can relate the length of the tail to the age of the surface of Listeria and make predictions as to the ratio of Listeria with varying tail lengths at a particular time after the initial infection. Since we know that division occurs about every 50 min, after 4 h we would predict that if we started with one Listeria in a macrophage, 16 bacteria would be found, two with long tails, two with medium tails, four with tiny tails, and eight with no tails or a ratio of 1:1:2:4. We measured the lengths of the tails on Listeria 4 h after infection in serial sections and confirmed this prediction. By decorating the actin filaments that make up the tail of Listeria with subfragment 1 of myosin we find (a) that the filaments are indeed short (maximally 0.3 microns in length); (b) that the filament length is approximately the same at the tip and the base of the tail; and (c) that the polarity of these filaments is inappropriate for myosin to be responsible or to facilitate movement through the cytoplasm, but the polarity insures that the bacterium will be located at the tip of a pseudopod, a location that is essential for spreading to an adjacent cell. Putting all this information together we can begin to unravel the problem of how the Listeria forms the cytoskeleton and what is the biological purpose of this tail. Two functions are apparent: movement and pseudopod formation.     

Effects of tail docking and docking length on neuroanatomical changes in healed tail tips of pigs

In pig production, piglets are tail docked at birth in order to prevent tail biting later in life. In order to examine the effects of tail docking and docking length on the formation of neuromas, we used 65 pigs and the following four treatments: intact tails (n=18); leaving 75% (n=17); leaving 50% (n=19); or leaving 25% (n=11) of the tail length on the pigs. The piglets were docked between day 2 and 4 after birth using a gas-heated apparatus, and were kept under conventional conditions until slaughter at 22 weeks of age, where tails were removed and examined macroscopically and histologically. The tail lengths and diameters differed at slaughter (lengths: 30.6±0.6; 24.9±0.4; 19.8±0.6; 8.7±0.6 cm; P<0.001; tail diameter: 0.5±0.03; 0.8±0.02; 1.0±0.03; 1.4±0.04 cm; P<0.001, respectively). Docking resulted in a higher proportion of tails with neuromas (64 v. 0%; P<0.001), number of neuromas per tail (1.0±0.2 v. 0; P<0.001) and size of neuromas (1023±592 v. 0 μm; P<0.001). The results show that tail docking piglets using hot-iron cautery causes formation of neuromas in the outermost part of the tail tip. The presence of neuromas might lead to altered nociceptive thresholds, which need to be confirmed in future studies.     

Bacteriophage SPO1 structure and morphogenesis. I. Tail structure and length regulation.

Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees. The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel. A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths. The tail length distribution suggests that the smallest integral length increment is a single disk of subunits. The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract. Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail. The implications of these findings with respect to tail length regulation are discussed.     

The use of spun columns in the purification of double-stranded cDNA appropriate for tailing

A reliable estimation of the efficiency of adding homopolymeric tails to double-stranded cDNA molecules by terminal deoxynucleotidyl transferase is extremely important in the construction of cDNA libraries. Appreciable differences in transformation efficiency result when the homopolymer tails to be annealed are of inadequate length. We report here that the use of a Sephadex G-50 spun column to remove oligo(dT)12-18 frequently coprecipitating in ethanol with the cDNA results in a more dependable estimation of tail lengths.     

Plasticity in courtship and sneaking behaviors depending on tail length in the male guppy, <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

Male guppies Poecilia reticulata exhibit two types of mating behavior, i.e., courtship displays for cooperative copulation and sneaking attempts for forced copulation. The frequencies of the two male mating behaviors are influenced by tail length. Males possessing long tails exhibit courtship displays less frequently and sneaking attempts more frequently than those possessing short tails, even though they have similar total lengths. To examine whether these male behavioral tendencies depending on tail length are genetically controlled or are determined by tail length per se, tail length manipulation was conducted. The tail lengths of males that had previously possessed longer tails were surgically shortened to a greater degree than those of their counterparts that had previously possessed shorter tails. Although the frequencies of the mating behaviors exhibited by the latter males did not apparently change, the former males clearly increased the frequency of courtship displays and decreased that of sneaking attempts following tail shortening. These results indicate that males adjust the frequencies of the two mating behaviors according to their tail length. Since females avoid cooperative mating with males possessing long tails, the change in mating behavioral patterns by males depending on their tail length may increase their mating opportunities.     

Patterns of morphometric variation in birds' tails: length, shape and variability

In Palaearctic birds, tail length was more variable than wing and tarsus, but not bill lengths. Tails of the ornamental shapes pin, lyre and graduated varied more in length than did non-ornamental shapes. Log coefficient of variation (C V) tail length showed an overall U-shaped relationship with longtailedness, but although the CV for most tail shapes increased in short-tailed species, only in ornamental shapes was C V also high in long-tailed species. C V of fork depth was lowest at a fork depth of 2, and considerably higher in shallow forked tails. CV streamer lengths were similar to CV deep fork depths. The more deeply forked tails thus seem ornamental. Phylogenetically independent contrasts confirmed in males, but not females, that long-tailed species had greater CV than medium-tailed species, and the greater CV of graduated than square tails, but the CV of short- and medium-tailed species did not differ. These comparisons, however, did not control for tail shape. The greater elongation and CV of tails with ornamental shapes are consistent with an influence of sexual/signal selection on these tails, and the increase in CV with longtailedness suggests that Weber's law applies to the perceptual threshold for tail length. Sexual selection may have a widespread, but moderate, influence on tail traits in birds.     

Extra-long bacteriophage T4 tails produced under in vitro conditions

Extra-long bacteriophage T4 tails have been produced under in vitro conditions from purified tails of normal length. These tails show a range of lengths suggesting that the basic increment of increased length is the 41 Å (Moody, 1971) axial repeating unit rather than the length of a normal tail. Some extra-long tails and tubes attached to baseplates show stain penetration down the central tunnel of the tube to approximately the normal tail length. The stain-penetrated tunnel, as visualised by three-dimensional reconstruction from the electron micrographs, has a diameter between 30 and 40 Å, sufficient to allow the passage of DNA. The exclusion of stain from the tunnel in the baseplate-near segment of the tube is interpreted as being due to the presence of additional material in the tunnel. The relevance of these observations to the assembly and length-regulation of the tail is discussed.     

The influence of natural selection and sexual selection on the tails of sea-snakes (Laticauda colubrina)

Many phenotypic traits perform more than one function, and so can influence organismal fitness in more than one way. Sexually dimorphic traits offer an exceptional opportunity to clarify such complexity, especially if the trait involved is subject to natural as well as sexual selection, and if the sexes differ in ecology as well as reproductive behaviour. Relative tail length in sea-snakes fulfils these conditions. Our field studies on a Fijian population of yellow-lipped sea kraits ( Laticauda colubrina ) show that relative tail lengths in male sea kraits have strong consequences for individual fitness, both via natural and sexual selection. Males have much longer tails (relative to snout-vent length) than do females. Mark-recapture studies revealed a trade-off between growth and survival: males with relatively longer tails grew more slowly, but were more likely to survive, than were shorter-tailed males. A male snake's tail length relative to body length influenced not only his growth rate and probability of survival, but also his locomotor ability and mating success. Relative tail length in male sea kraits was thus under a complex combination of selective forces. These forces included directional natural selection (through effects on survival, growth and swimming speed) as well as stabilizing natural selection (males with average-length tails swam faster) and stabilizing sexual selection (males with average-length tails obtained more matings). In contrast, our study did not detect significant selection on relative tail length in females. This sex difference may reflect the fact that females use their tails primarily for swimming, whereas males also must frequently use the tail in terrestrial locomotion and in courtship as well as for swimming.     

Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.     

Tail position affects the body temperature of rats during cold exposure in a low-energy state

Rats place their tails underneath their body trunks when cold (tail-hiding behavior). The aim of the present study was to determine whether this behavior is necessary to maintain body temperature. Male Wistar rats were divided into ‘fed’ and ‘42-h fasting’ groups. A one-piece tail holder (8.4 cm in length) that prevented the tail-hiding behavior or a three-piece tail holder (2.8 cm in length) that allowed for the tail-hiding behavior was attached to the tails of the rats. The rats were exposed to 27°C for 180 min or to 20°C for 90 min followed by 15°C for 90 min with continuous body temperature and oxygen consumption measurements. Body temperature decreased by −1.0 ± 0.1°C at 15°C only in the rats that prevented tail-hiding behavior of the 42-h fasting group, and oxygen consumption increased at 15°C in all animals. Oxygen consumption was not different between the rats that prevented tail-hiding behavior and the rats that allowed the behavior in the fed and 42-h fasting groups under ambient conditions. These results show that the tail-hiding behavior is involved in thermoregulation in the cold in fasting rats.     

Apigenin ameliorates gamma radiation-induced cytogenetic alterations in cultured human blood lymphocytes

The aim of the present study was to assess the protective effect of apigenin, a dietary flavone, against cytogenetic alterations in human peripheral blood lymphocytes (HPBL) induced by Cobalt-60 radiation (3Gy). Results of MTT [3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay revealed that 37.2μM of apigenin was found to be non-toxic in HPBL. At this dose (37.2μM) of apigenin, the LD(50) radiation dose of HPBL increased from 2.9Gy to 3.4Gy, which resulted in a DMF of 1.17. Apigenin (37.2μM) treatment 1h before irradiation significantly (p<0.05) reduced DNA damage in irradiated HPBL as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment). Moreover, apigenin treatment significantly decreased the frequencies of dicentric (DC), acentric fragments (AF), and acentric rings (AR) in irradiated HPBL. Apigenin pretreatment also reduced the radiation-induced CBMN (cytokinesis blocked micronuclei) anomalies such as micronuclei (MNi), nucleoplasmic bridges (NPB) and nuclear buds (NBUD) in HPBL. These results also showed that there was a significant correlation between NPB and DC frequencies and MNi and AF+AR. Treatment with apigenin alone had no significant effect on DNA damage and chromosomal aberrations in HPBL. Thus, the current studies indicate that apigenin protects HPBL from radiation-induced cytogenetic alterations.     

Tail length in birds in relation to tail shape,general flight ecology and sexual selection

Relative tail length (longtailedness) of Palearctic birds was assessed by the standardized residuals of log–log regressions of tail length on wing length and tarsus length. The mean degree of tail shortening was greater than mean degree of tail lengthening, but there was a greater frequency of extreme long-tailed than short-tailed species. Longtailedness was greater in ornamental pin, lyre, deep forked and graduated shaped tails. These shapes (except graduated, for which data were lacking) were also relatively long-tailed according to shortest-rectrix lengths, this extra length potentially contributing compensatory lift. In forked tails, tail ratio increased linearly with longtailedness to above the aerodynamic optimum, and thus the most elongated forked tails were also more deeply forked. Tail shortening was marked for rounded tails, a surprising result in view of their slightly ornamental shape. Phylogenetically independent contrasts showed significantly greater longtailedness in graduated than square-tailed species, confirming the species-wide analysis. In phylogenetically independent contrasts of longtailedness and ecological factors, short-tailed species had significantly greater flight distances than medium-tailed species, but long- and medium-tailed species did not differ in migratory distance, foraging distance, overall flight distance or importance of aerial foraging. The data suggest that ecological factors, i.e. natural selection, are more important in the evolution of short-tailedness than longtailedness in birds, and that an additional influence of sexual selection on tail length and shape is also widespread.     

The influence of X-rays on human erythrocytes. Primary radicals

The effects on human erythrocytes of water-derived radicals generated by X-rays were studied under anaerobic conditions and in the presence of oxygen. Erythrocyte damage was estimated on the basis of the reduced GSH and MetHb content in the erythrocytes, the -SH group content in the membrane proteins and the amount of K(+)released from the erythrocytes. The results obtained show that the level of reduced GSH was the most sensitive indicator of erythrocyte damage by X-rays followed by the efflux of K(+). The processes of GSH oxidation took place most rapidly under air. At a dose of 100 Gy, the level of GSH fell to about 50%, whereas under argon and N(2)O to about 75% and 65%, respectively. A slight increase in the efflux of K(+)was observed in preparations irradiated under air. However, when erythrocytes were irradiated under argon and N(2)O, the loss of K(+)occurred at a dose 8-times higher. Changes in the remaining parameters occurred at considerably higher doses. On the basis of the results obtained one can say that oxygen is a factor increasing the toxicity of(.)OH radicals towards erythrocytes; however, e(-)(aq)present in the system can cause a decrease in damage to certain cellular components.     

The relationship between o.e.r., r.b.e. and number of fractions for X-ray- or neutron-induced skin damage.

The skin reactions in aerated and hypoxic mouse tails after single or fractionated doses of 250 kV X-rays or fast neutrons (6 MeV deuterons on beryllium) have been measured. The o.e.r. for one to sixteen fractions of X-rays remains constant, while that for one to ten fractions of neutrons decreases with increasing neutron fractionation and decreasing neutron dose/fraction. The o.e.r. for X-rays was 1.7, for single-neutron doses 1.4, and for ten fractions of neutrons 1.25. It was anticipated that the o.e.r. for neutron-induced damage would decrease further as neutron fractionation is increased because the contribution to damage from the highest LET components of dose, the alpha and heavy recoil particles, would increase relative to the lowe LET components. The r.b.e. values obtained for skin damage were higher at all neutron doses/fraction examined in this study on tails than all those previously obtained in studies on skin at other sites on four species. This may be due to the influence of hypoxia on the r.b.e. measurements in the mouse tail.     

Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for "Chi" recognition complexes

The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3'-single-stranded (ss) DNA ((dT)n) tails was studied. These studies were designed to test a previous proposal that the 3'-(dT)n tail can be looped out upon binding RecBC and RecBCD for 3'-ssDNA tails with n>or=6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3'-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3'-(dT)n tail. However, placing PEG at the end of the 3'-(dT)n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3'-(dT)6 tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecB(Deltanuc)C, suggest that looping of the 3'-tail (when n>or=6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD-DNA complexes suggests that the loop in the 3'-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3'-ssDNA tail might form upon encounter of a "Chi" recognition sequence during unwinding of DNA by the RecBCD helicase.     

Measurement of tumor oxygenation using new frequency domain phosphorometers

Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.