Distribution and Chromatographic Characterisation of Gastrin and Cholecystokinin in the Rat Central Nervous System

Abstract: Two tissue extraction techniques and two radioimmunoassays were used to study the distribution of gastrin and cholecystokinin in rat brain. Small amounts of gastrin were found in extracts of neurohypophysis, but in neither ice-cold 90% methanol nor in boiling water-acetic acid extracts of the other 33 brain areas studied. Cholecystokinin was found in equivalent amounts in both types of extract of 31 areas. The distribution was similar to that in previous studies. The components of cholecystokinin immunoreactivity were characterised in 10 rat CNS tissues using four tissue extraction methods in conjunction with gel filtration and ion-exchange chromatography. The results demonstrated that gastrins were present only in the neurohypophysis and that in all other rat CNS tissues the main molecular component was indistinguishable from the sulphated octapeptide of cholecystokinin. Minor immunoreactive components were observed in all types of extract of all tissues with the properties of the desulphated octapeptide and the C-terminal tetrapeptide amide, suggesting they are genuine tissue components, not extraction artefacts. Large molecular forms of cholecystokinin were not detected in any tissue. The results emphasise the necessity of using two or more extraction methods and two or more chromatography systems in such a study.     

The Cytoplasmic pH Influences Hyphal Tip Growth and Cytoskeleton-Related Organization

The regulatory role of protons in hyphal tip growth was investigated by using membrane-permeant weak acids to acidify cytoplasm of the oomycete Saprolegnia ferax. Acetic acid decreased cytoplasmic pH from approximately pH 7.2 to 6.8, as shown by SNARF-1 measurements of cytoplasmic pH. Inhibition of growth in a dose-dependent manner by acetic, propionic, and isobutyric acid was accompanied by changes in positioning and morphology of mitochondria and nuclei, condensation of chromatin, disruptions in peripheral actin, and increases in hyphal diameter. These cellular alterations were fully reversible, and during recovery, major cytoplasmic movements and extensive apical vacuolations were observed. The results are consistent with proton regulation of the cytoskeleton, nuclear matrix, and/or chromosomes. However, a macroscopic cytoplasmic gradient of H+ in hyphae was not revealed by SNARF-1, indicating that if such a H+ gradient were required, it must occur at a finer level than we detected.     

Comparison of three commercial vaccines for preventing persistent infection with bovine viral diarrhea virus

Eighty crossbred beef heifers were randomly allocated to four groups to evaluate the efficacy of vaccination in preventing development of calves persistently infected with bovine viral diarrhea virus (BVDV). Group 1 (n = 11) was non-vaccinated controls, whereas three groups were vaccinated with commercially available multivalent BVDV vaccines at weaning (∼7 mo of age), 28 d post-weaning, ∼1 y of age, and 28 d later. Groups 2 (n = 23) and 3 (n = 23) were given a modified-live BVDV vaccine, whereas Group 4 was given an inactivated BVDV vaccine. Heifers were bred by AI and subsequently exposed to two bulls. At 61 d after AI, 70 heifers were pregnant (n = 10 for Group 1 and n = 20/group for Groups 2, 3, and 4). Three cattle persistently infected with BVDV were commingled with the pregnant heifers (in an isolated pasture) from 68 to 126 d after AI. Thereafter, viremias were detected in pregnant heifers from Groups 1, 3, and 4 (10/10, 1/20, and 10/20, respectively), but not in pregnant heifers from Group 2 (0/20). Resulting calves were assessed for persistent infection using serum PCR, ear notch antigen capture-ELISA, and immunohistochemistry. Persistently infected calves were only produced in Group 1 (10/10) and Group 4 (2/18). In conclusion, commercial vaccines provided effective fetal protection despite prolonged natural exposure to BVDV. Given that viremias were detected in 11 vaccinated heifers after BVDV exposure, and two vaccinated heifers gave birth to persistently infected calves, there is continued need for biosecurity and diagnostic surveillance, in addition to vaccination, to ensure effective BVDV control.     

Effects of Opioid Peptides Containing the Sequence of Met5-Enkephalin or Leu5-Enkephalin on Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.     

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.     

A non‐canonical role of the p97 complex in RIG‐I antiviral signaling

RIG‐I is a well‐studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4‐Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG‐I. The p97 complex is able to directly bind both non‐ubiquitinated RIG‐I and the E3 ligase RNF125, promoting K48‐linked ubiquitination of RIG‐I at residue K181. Viral infection significantly strengthens the interaction between RIG‐I and the p97 complex by a conformational change of RIG‐I that exposes the CARDs and through K63‐linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG‐I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.     

Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.     

Low-resolution structural studies of human Stanniocalcin-1

Background  

Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di- or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC₅₀ and "big STC", which molecular weights range from 56 to 135 kDa.     

Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm

Background  

Marker Assisted Selection (MAS) is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials.