Inferring diet, feeding behaviour and causes of mortality from prey-induced injuries in a New Zealand fur seal.

New Zealand fur seals Arctocephalus forsteri are the most abundant of the 4 otariid (eared seal) species distributed across Australasia. Analyses of stomach contents, scats and regurgitates suggest a diet dominated by bony fish and squid, with cartilaginous species (e.g. sharks and rays) either absent or underrepresented because of a lack of preservable hard parts. Here we report on a subadult specimen from south-eastern Australia, which was found ashore emaciated and with numerous puncture wounds across its lips, cheeks, throat and the inside of its oral cavity. Fish spines embedded in the carcass revealed that these injuries were inflicted by chimaeras and myliobatiform rays (stingrays and relatives), which matches reports on the diet of A. forsteri from New Zealand, but not South Australia. Shaking and tearing of prey at the surface may help to avoid ingestion of the venomous spines, perhaps contributing to their absence from scats and regurgitates. Nevertheless, the number and severity of the facial stab wounds, some of which led to local necrosis, likely affected the animal's ability to feed, and may account for its death. Despite their detrimental effects, fish spine-related injuries are difficult to spot, and may be a common, albeit cryptic, type of trauma. We therefore recommend that stranded seals be systematically examined for this potentially life-threatening pathology.

The biomechanical consequences of longirostry in crocodilians and odontocetes.

Unrelated clades of aquatic tetrapod have evolved a similar range of skull shapes, varying from longirostrine (elongate and narrow rostrum) to brevirostrine (short rostrum). However, it is unclear which aspects of organismal performance are associated with this convergence in the range of skull shapes. Furthermore, it is not known how fundamental anatomical differences between groups influence these relationships. Here we address this by examining the load bearing capabilities of the skulls of two of the most diverse groups of living aquatic tetrapod: crocodilians and odontocetes. We use finite element analysis to examine the abilities of different cranial morphologies to resist a range of biologically relevant feeding loads including biting, shaking and twisting. The results allow for form/function relationships to be compared and contrasted between the two groups. We find that cranial shape has similar influences on performance during biting, shaking or twisting load cases at the anterior tooth positions, e.g. brevirostrine species experienced less strain than longirostrine species. The pattern of this form/function relationship is similar for both crocodilians and odontocetes, despite their fundamentally different anatomies. However, when loading teeth at the posterior end or middle of the tooth row the results do not follow the same pattern. Behavioural differences in bite location plays a key role in determining functional abilities in aquatic tetrapod taxa.

Growth factors differentially regulate neuronal Cav channels via ERK-dependent signalling.

Voltage-gated calcium channels (Ca(v)) are tonically up-regulated via Ras/extracellular signal-regulated kinase (ERK) signalling in sensory neurones. However, the mechanisms underlying the specificity of cellular response to this pathway remain unclear. Neurotrophic factors are attractive candidates to be involved in this process as they are key regulators of ERK signalling and have important roles in neuronal survival, development and plasticity. Here, we report that in rat dorsal root ganglion neurones, endogenous nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) and epidermal growth factor (EGF) are all involved in tonic ERK-dependent up-regulation of Ca(v) channels. Chronic (overnight) deprivation of growth factors inhibits total Ca(v) current according to developmental changes in expression of the cell surface receptors for NGF, GDNF and EGF. Whilst EGF specifically regulates transcriptional expression of Ca(v)s, NGF and GDNF also acutely modulate Ca(v) channels within a rapid ( approximately 10min) time-frame. These acute effects likely involve changes in the biophysical properties of Ca(v)s, including altered channel gating rather than changes in surface expression. Furthermore, NGF, GDNF and EGF differentially regulate specific populations of Ca(v)s. Thus, ERK-dependent regulation of Ca(v) activity provides an elegant and extremely flexible system with which to tailor calcium influx to discrete functional demands.

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels.

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.

Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway.

The small G-protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage-dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co-expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs). Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41 % compared with control cells (GFP alone). In contrast, a farnesylation-defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors), significantly reduced calcium current density by 47 %. Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 alpha-subunit of phosphatidylinositol 3-kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf-1 and the mitogen-activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67 %, suggesting that Raf-1 activation is sufficient for Ras enhancement of calcium current in these cells. Raf-1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45 % after 10-15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras-MAPK pathway rather than gene expression-mediated effects. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- ( approximately 40 %) and L- ( approximately 40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre-treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.

cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS.

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.

Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras.

Influx of calcium through voltage-dependent calcium channels (VDCCs) has been implicated in the processes of cell growth and differentiation. Various signalling proteins, including nerve growth factor (NGF), p21-ras and src tyrosine kinases, have been suggested to have a role in the regulation of neuronal VDCCs. Using the whole-cell patch-clamp technique we have investigated the role of endogenous p21-ras in the regulation of VDCCs in primary cultured dorsal root ganglion (DRG) neurons obtained from neonatal rats. Neutralization of endogenous p21-ras by microinjection of p21-ras antibody (Y13-259) reduced the maximum peak barium current, I(max), whereas microinjection of oncogenic p21-K-ras increased the current. Thus, endogenous p21-ras is involved in the tonic regulation of calcium currents in these cells. Intracellular application of a phosphopeptide, Trk 490, which prevents the binding of the adaptor protein shc to the activated NGF receptor, so blocking p21-ras activation, reduced I(max). Similarly, deprivation of NGF by overnight incubation in NGF-free medium also reduced I(max). Together, these results suggest that NGF receptor tyrosine kinase activation of p21-ras is likely to be involved in the tonic regulation of VDCCs in DRG neurons. Deprivation of NGF combined with microinjection of p21-ras antibody (Y13-259), however, caused an even greater reduction of I(max). Thus, NGF activation can only partially explain the regulation of these currents by endogenous p21-ras. Src tyrosine kinases have been suggested to activate p21-ras. In DRG neurons, microinjection of purified src tyrosine kinase, pp60c-src, increased I(max) in these cells. However, co-microinjection of pp60c-src with Y13-259 antibody prevented the increase in I(max), implying that pp60c-src can also regulate calcium currents via the activation of endogenous p21-ras. Further support for the involvement of tyrosine kinases in VDCC regulation was provided by the application of the general tyrosine kinase inhibitor, genistein, which also reduced I(max). Thus, VDCCs in rat DRG neurons appear to be tonically up-regulated by endogenous p21-ras. This effect appears largely to involve NGF receptor tyrosine kinase activation of p21-ras. In addition, src tyrosine kinase may also regulate VDCCs, possibly via p21-ras.

Spectral plasticity of H1 horizontal cells in carp retina: independent modulation by dopamine and light-adaptation.

It was shown previously that the spectral sensitivity of luminosity/H1-type horizontal cells (HCs) in carp retinae reflects the absorption spectrum of red-sensitive cones for long wavelengths but can appear highly variable and "truncated' in the short-wavelength region of the spectrum. We have found that light-adaptation sharpened the red-sensitive spectral peak and decreased the blue/red response amplitude ratio (B/R ratio), mainly by decreasing the response to short-wavelength stimuli. The adaptation effect was more pronounced for red background light than for blue. During dark adaptation, the B/R ratio increased steadily. Exogenous dopamine (DA; 5 microM) changed the spectral response profile in a similar way to light-adaptation. However, the effect of light-adaptation in reducing the B/R ratio was still seen in retinae bathed in 5 microM DA. This effect of background adaptation was also recorded in retinae bathed in 37 microM haloperidol, as well as in retinae pretreated with 6-hydroxydopamine (i.e. DA-depleted). The results suggest that (i) short-wavelength-sensitive cones play a dynamic role in determining the spectral response profile of H1 HCs and (ii) spectral response characteristics are modulated independently by exogenous DA and an unknown endogenous neuromodulator which is activated by light-adaptation.

Sensitivity of valinomycin-based K(+)-selective micro-electrodes to inhibitors of K+ transport.

The sensitivies of double-barrelled K(+)-selective micro-electrodes (KSMs) employing the low-impedance membrane cocktail based on the neutral K(+)-selective ion carrier valinomycin (Fluka, Cocktail B 60398) to the following 3 different classes of inhibitors of K+ transport were measured: (1) general metabolic inhibitors (dinitrophenol, potassium cyanide, sodium azide, rotenone, dicyclohexylcarbodiimide, salicylhydroxamic acid); (2) P-type ATPase inhibitors (vanadate, ouabain, amiloride, SCH 28080); and (3) anion-dependent K+ transport inhibitors (bumetanide, 4-acetamide-4-isothiocyanostilbene-2,2-disulphonic acid). Of the 12 inhibitors tested, only dinitrophenol had any significant effect on the response of KSMs to K+ activity. Comparison of the calibrations in solutions with and without 0.1 mM dinitrophenol showed that this inhibitor behaved as a 'classical' interferent whereby its contribution to the K+ activity signal was statistically significant at K+ activities of 36.0 mM and less. However, at higher K+ activities (97.0 mM), dinitrophenol interference was not significant. It was possible to correct for the DNP interference and to obtain measurements of intracellular K+ activity in insect muscles.

Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta-subunit in rat neurones.

1. The beta-subunit has marked effects on the biophysical and pharmacological properties of voltage-dependent calcium channels. In the present study we examined the ability of the GABAB agonist (-) -baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta-subunit immunoreactivity, 108-116 h after microinjection of a beta-subunit antisense oligonucleotide. 2.We observed that, although the calcium channel current was markedly reduced in amplitude following beta-subunit depletion, the residual current (comprising both N- and L-type calcium channel currents) showed an enhanced response to application of (-) -baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta-subunit for binding to the calcium channel alpha 1-subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta-subunit, resulting in increased inhibition. 3. This hypothesis is supported by evidence that an antibody against the calcium channel beta-subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S-(-) -Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha-subunit (G alpha o) with its calcium channel effector which may operate as a GTPase-activating protein. 4. These data suggest that the calcium channel beta-subunit when complexed with the beta 1-subunit normally inhibits its association with activated G(o). It may function as a GTPase-activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (-) -baclofen.

Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels.

1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta- with alpha 1-subunits in oocytes and other expression systems. 4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha 1-subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.

Adaptive behavioral responses to potential infertility among survivors of testis cancer.

In a retrospective study of 153 testis cancer survivors, we examined the sociodemographic and clinical determinants of attitudes and behaviors toward illness-induced infertility. Five fertility adjustment responses were identified: sperm-banking awareness (SBA); adoption awareness (AA); fertility testing (FT); trying to father children (TFC); and fertility distress (FD). Although responses to infertility are multidetermined, these data demonstrate there is a distinct sociodemographic and clinical profile for the subgroups of men who engage in different fertility-related behaviors. Multivariate analysis results show that men most likely to be concerned with banking sperm are those who at diagnosis are younger (less than 35 years), childless, college educated, and whose relationships have become strained. Men who sought fertility tests were childless, college graduates, and able to ejaculate. The only factor predicting adoption was childlessness. Those married men attempting to father children were also less than 35 years of age at diagnosis and without ejaculatory dysfunction. The men at greatest risk for continued distress about infertility were those who remained childless and had posttreatment ejaculatory dysfunction. Residual infertility distress also was significantly associated with treatments that included extensive retroperitoneal lymph node dissection (RPLND) surgery, indicating that the latter is a "risk factor" in survivors' long-term distress. These data, while not definitive, show that the prerogative to have children is very important to men and that losing it sets into motion a range of both adverse emotions and adaptive responses. Adjustment to infertility is a complex process that begins at diagnosis and extends long after treatment is completed.

Psychosocial factors, curative therapies, and behavioral outcomes. A comparison of testis cancer survivors and a control group of healthy men.

In a retrospective study of 223 testis cancer survivors and 120 controls matched sociodemographically, we examined the relative impact of sociodemographic and clinical factors on long-term outcomes in the areas of sexual function, relationships, employment, and mental outlook. For most of the survivors, testis cancer did not lead to unemployment (4.5%), divorce (6.8%), or disabling psychological problems. Multivariate analysis results confirm that cancer survivors report significantly more infertility and sexual performance distress, but not more desire distress, than the control group. Survivors' sexual impairment varied according to treatment received (and therefore histologic factors) and sociodemographic variables. Parental status (not having children) and education (college or less) independently predict infertility distress, whereas education and lower occupational level independently predicted sexual performance distress. Adjusting for socioeconomic status (SES), the men with advanced testis cancer who received chemotherapy and standard retroperitoneal lymph node dissection (RPLND) had significantly more infertility and performance distress than those men who received other treatments. Neither the treatment or SES variables predicted disrupted relationships or a deteriorated mental outlook. However, men with sexual impairment distress were more likely to report strained relationships and a pessimistic mental outlook. These findings have implications for treatment decisions and can be used to identify subgroups of survivors who could benefit from counseling and sexual rehabilitation services.

Lack of evidence for gastrointestinal control of sodium excretion in unanesthetized rabbits.

Experiments were performed to determine whether unanesthetized rabbits exhibit a greater natriuresis after oral administration of a hypertonic sodium chloride solution (1.5 mmol NaCl/kg) than after the intravenous administration of the same solution. Male rabbits (New Zealand) were placed on a low sodium diet for 4 days, and on the fifth day a hypertonic NaCl solution (616 mM) was given either by stomach tube (GI) or intravenously (IV), while an equal volume of a hypotonic NaCl solution (31 mM) was given by the alternate route. The studies were repeated on each rabbit so that paired observations were obtained. No differences in plasma Na concentration, peripheral hematocrit, or urinary sodium excretion were observed between the GI and IV groups after administration of the hypertonic NaCl solution. In seven rabbits that were permitted access to food and water following NaCl administration by either route, urinary sodium excretion tended to be reduced (P = 0.08), but, again, no significant differences in plasma sodium concentration, peripheral hematocrit, or urinary sodium excretion were observed between the GI and IV routes of sodium administration. Accordingly, we could find no evidence to support the existence of a GI or portal Na receptor system that regulates urinary sodium excretion in the unanesthetized rabbit.