Ligand-binding characteristics of feline insulin-binding immunoglobulin G

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e⁻⁴ M for fraction B (low affinity IgGs) and 2e⁻⁵ M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.     

Inhibition of transient receptor potential vanilloid type 1 through α2 adrenergic receptors at peripheral nerve terminals relieves pain

The activation of α₂ adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α₂ receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α₂ receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α₂ receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α₂ receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α_2C receptor mRNA quantified by real-time PCR was highest followed by those of α_2A and α_2B receptors in DRGs. α_2A and α_2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α₂ receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia.     

A possible role of central serotonin in L‐tryptophan‐induced GH secretion in cattle

To clarify the role of serotonin (5‐HT) in the regulatory mechanism of L‐tryptophan (TRP)‐induced growth hormone (GH) secretion in cattle, changes in 5‐HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1‐h intervals. The concentration of 5‐HT in CSF was determined by high‐performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio‐immunoassay and enzyme‐immunoassay. Concentrations of 5‐HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.     

Effects of adding food by‐products mainly including noodle waste to total mixed ration silage on fermentation quality,feed intake,digestibility, nitrogen utilization and ruminal fermentation in wethers

Four wethers were used in a 4 × 4 Latin square design experiment to evaluate the applicability of two types of total mixed ration (TMR) silage with food by‐products. Four food by‐products (i.e., potato waste, soy sauce cake, soybean curd residue and noodle waste) were obtained and mixed with commercial concentrate (CC) as TMR silage. The two types of TMR silage, T1 and T2, each contained CC, in addition to all by‐products for T1 (TRE1), and soy sauce cake and noodle waste for T2 (TRE2) on a dry matter (DM) basis. The silage was well‐fermented with low pH values and high lactic acid concentration. As the experimental treatments, T1, T2 and CC (CTL) were fed with a basal diet. The result showed that the digestibility of DM and organic matter (OM) were higher for T1 than for CC (P < 0.05), while crude protein digestibility was not significantly different among T1, T2 and CC. The retained nitrogen was not affected by inclusion of food by‐products. Ruminal pH in TRE1 and TRE2 immediately decreased compared to that in CTL. The study suggested that the two types of TMR silage, including food by‐products, might be used as a substitute for commercial concentrate.     

Effect of intracerebroventricular injections of prolactin‐releasing peptide on prolactin release and stress‐related responses in steers

Some evidence suggests that there might be a species difference in the effect of intracerebroventricularly administered (ICV) prolactin‐releasing peptide (PrRP) between rodents and sheep. We compared the levels of cortisol (CORT) and prolactin (PRL), rectal temperature (RT) and behavioral responses to ICV bovine PrRP (bPrRP) in steers. ICV bPrRP (0.2, 2 and 20 nmol/200 µL) tended to evoke a dose‐related increase in CORT concentrations and 0.2 nmol of bPrRP induced transient increase in PRL concentrations. A significant time–treatment interaction was observed for the percent change of CORT (P < 0.05) and PRL (P < 0.05) from pre‐injection value. The time–treatment interaction for changes in RT was not significant (P = 0.50). There tended to be a difference among the four treatments in terms of maximum change in RT from the pre‐injection value between 0 and 90 min (P < 0.1). Stress‐related behavioral signs were not observed in the present experiment. These findings indicate that ICV bPrRP increased CORT and PRL levels, suggesting that central PrRP might participate in controlling the hypothalamo‐pituitary‐adrenal axis and PRL release in cattle, unlike sheep. In contrast, central PrRP is unlikely to be involved in controlling the behavior of this species because ICV bPrRP did not induce marked changes in their behavior.