Detyrosination of tubulin is not correlated to cold-adaptation of microtubules in cultured cells from the Atlantic cod (Gadus morhua)

Summary Isolated cod brain microtubules from the cold-adapted Atlantic cod (Gadus morhua) have previously been shown to be highly detyrosinated, a post-translational modification of tubulin usually found in stable subsets of microtubules. In this study we found this was not restricted only to isolated brain microtubules. Microtubules in primary cultures of brain and skin cells were composed of both tyrosinated (Tyr)- and detyrosinated (Glu)-tubulin seen by immunocytochemistry. Immunoelectron microscopy of isolated microtubules showed that individual microtubules were composed of a mixture of Tyr- and Glu-tubulin. Leukocytes with extending lamellopodia contained only microtubules stained with the antibody against Tyr-tubulin, and isolated heart tubulin lacked both Tyr- and Glu-tubulin, suggesting that a relative high level of detyrosination is a characteristic of most, but not all, cod microtubules. Brain cell microtubules were more resistant to mitotic inhibitors than skin cell microtubules, but this was not correlated to a difference in detyrosination. Brain and skin cell microtubules were only partially disassembled when incubated at 0°C. Upon reassembly of microtubules at 12°C, microtubules were still made of mixtures of Tyr- and Glu-tubulin, indicating that detyrosination of assembled microtubules is rapid and/or that in cod cells, in contrast to mammalian cells, Glu-tubulin can reassemble to microtubules. Our data show that most cod microtubules are highly detyrosinated, but this is not the cause of their cold adaptation or drug stability.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

Highly sensitive method for the determination of melatonin by normal-phase high-performance liquid chromatography with fluorometric detection

In the present study a new chromatographic method was developed to quantify melatonin in rat pineal that can be extended to other tissues. Melatonin was extracted from an acid homogenate with ethyl acetate to avoid amine interference. HPLC was performed with silica normal-phase column and fluorescence detection. This method is sensitive enough for detecting melatonin in a single pineal gland with a detection limit of 3 pg/mg tissue.     

Isolation of gonadotrops from the pituitary of the African catfish,Clarias lazera

Summary Dispersed pituitary cells from male African catfish, Clarias lazera, were fractionated in a density gradient of Percoll. Five fractions were isolated, consisting of about 6, 19, 39, 95 and 83% gonadotrops, respectively. The gonadotrops were identified by their ultrastructural characteristics, by immunocytochemistry, and by measuring their hormone content. After one day in culture, in each fraction the secretion of gonadotropin could be stimulated by a luteinizing hormone-releasing hormone analogue, indicating that the cells had retained their functional integrity. Since the regulatory mechanisms of different cell types from the pituitary have some similarity, purification of the gonadotrops provides a model to study the regulation of gonadotropin secretion.A portion of the results was presented as a poster at the XII^th Conference of the European Society of Comparative Endocrinology, Sheffield, July 31–August 5, 1983     

Identification of luteinizing hormone-releasing hormone (LH-RH) in the brain and pituitary gland of a fish by immunocytochemistry.

For the first time immunoreactive luteinizing hormone-releasing hormone (LH-RH) is demonstrated in both the brain and pituitary gland of a teleost (Xiphophorus maculatus) using an immunoperoxidase procedure. It is specifically localized in the perikarya and their axons of the ventral telencephalon and nucleus lateralis tuberis and within and between the gonadotrops and within some cells of the pars intermedia. These immunoreactions are extinguished when antiserum to LH-RH is preincubated with LH-RH antigen but not with neurohypophysial hormones.     

LHRH-immunoreactive cells in the brain of the three-spined stickleback,Gasterosteus aculeatus L. (Gasterosteidae)

Summary Cells immunoreactive with an anti-LHRH serum were visualized in the brain of the three-spined stickleback, Gasterosteus aculeatus, by means of the PAP technique. Positive cells were found in a periventricular position in the nucleus praeopticus pars magnocellularis, the nucleus dorsomedialis thalami, the nucleus ventromedialis thalami, the nucleus periventricularis posterior, and in the periventricular dorsomedian tegmentum. These cells were frequently observed to contact the CSF.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Early host–microbe interaction in a peri‐implant oral mucosa‐biofilm model

The host‐microbe relationship is pivotal for oral health as well as for peri‐implant diseases. Peri‐implant mucosa and commensal biofilm play important roles in the maintenance of host‐microbe homeostasis, but little is known about how they interact. We have therefore investigated the early host‐microbe interaction between commensal multispecies biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Porphyromonas gingivalis) and organotypic peri‐implant mucosa using our three‐dimensional model. After 24 hr, biofilms induced weak inflammatory reaction in the peri‐implant mucosa by upregulation of five genes related to immune response and increased secretion of IL‐6 and CCL20. Biofilm volume was reduced which might be explained by secretion of β‐Defensins‐1, ‐2, and CCL20. The specific tissue reaction without intrinsic overreaction might contribute to intact mucosa. Thus, a relationship similar to homeostasis and oral health was established within the first 24 hr. In contrast, the mucosa was damaged and the bacterial distribution was altered after 48 hr. These were accompanied by an enhanced immune response with upregulation of additional inflammatory‐related genes and increased cytokine secretion. Thus, the homeostasis‐like relationship was disrupted. Such profound knowledge of the host‐microbe interaction at the peri‐implant site may provide the basis to improve strategies for prevention and therapy of peri‐implant diseases.     

A layover in Europe: Reconstructing the invasion route of asexual lineages of a New Zealand snail to North America

Non‐native invasive species are threatening ecosystems and biodiversity worldwide. High genetic variation is thought to be a critical factor for invasion success. Accordingly, the global invasion of a few clonal lineages of the gastropod Potamopyrgus antipodarum is thus both puzzling and has the potential to help illuminate why some invasions succeed while others fail. Here, we used SNP markers and a geographically broad sampling scheme (N = 1617) including native New Zealand populations and invasive North American and European populations to provide the first widescale population genetic assessment of the relationships between and among native and invasive P. antipodarum. We used a combination of traditional and Bayesian molecular analyses to demonstrate that New Zealand populations harbour very high diversity relative to the invasive populations and are the source of the two main European genetic lineages. One of these two European lineages was in turn the source of at least one of the two main North American genetic clusters of invasive P. antipodarum, located in Lake Ontario. The other widespread North American group had a more complex origin that included the other European lineage and two New Zealand clusters. Altogether, our analyses suggest that just a small handful of clonal lineages of P. antipodarum were responsible for invasion across continents. Our findings provide critical information for prevention of additional invasions and control of existing invasive populations and are of broader relevance towards understanding the establishment and evolution of asexual populations and the forces driving biological invasion.     

In vitro propagation, histochemistry, and analysis of essential oil from conventionally propagated and in vitro-propagated plants of Varronia curassavica Jacq

Varronia curassavica is cultivated for the production of an essential oil useful in the pharmaceutical industry for its strong anti-inflammatory effect. Despite a growing demand, only a few studies have evaluated alternative sources of obtaining plantlets or ways to increase the yield of essential oil from this species. Therefore, this study aimed to optimize the in vitro multiplication rate and analyze the histochemistry and sesquiterpene production potential of conventionally propagated V. curassavica plants, in vitro shoots, and acclimatized plants derived from in vitro shoots. For axillary bud proliferation, Murashige and Skoog medium was supplemented with 6-benzyladenine and thidiazuron alone or in combination with naphthalene acetic acid. Axillary bud proliferation was obtained from culture of nodal or apical segments on medium containing half-strength Murashige and Skoog salts without growth regulators. After 35 d of culture, an average of five buds developed per explant. Elongation and rooting of shoots also occurred in this medium. After the transfer of rooted plants to ex vitro conditions, 100% of the plantlets survived. Histochemical analysis of leaf tissue showed the presence of lipids, acidic lipids, essential oil, phenols, and flavonoids. The essential oils from conventionally propagated and acclimatized plants were extracted by hydrodistillation and analyzed using gas chromatography. The essential oil from acclimatized plants had a similar profile to that from ex vitro plants, but with a higher concentration of the anti-inflammatory compound alpha-humulene.