A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system. (C) 2010 Elsevier Ltd. All rights reserved.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [

Steroid 21-hydroxylase expression in cultured rat astrocytes

Over the past decade or so it has become widely recognised that the brain is a significant steroidogenic organ. Many publications have highlighted the ability of the brain to synthesise and interconvert a large number of steroid products including cholesterol, progesterone and testosterone. In this study, in vitro experiments were performed to determine if 21-hydroxylation of steroids is undertaken by rat brain astrocytes in culture. This is a common reaction that occurs in the adrenal gland and other organs in mammals, catalysing the conversion of pregn-4-ene-3,20-dione (progesterone) to 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone).

Previous reports have indicated that 21-hydroxylation occurs within the rat brain, however, the precise identity of the cells expressing 21-hydroxylase has not yet been determined. Several metabolites, such as 5α-pregnan-3α-ol-20-one (tetrahydroprogesterone) and 3α,21-dihydroxy-5-pregnan-20-one (tetrahydrodeoxycorticosterone) were of particular interest because of their modulatory role in neuronal function, such as their agonist activity at γ-aminobutyric acid (GABA_A) receptors.

Evidence was obtained for the expression of peripheral 21-hydroxylase enzyme (P450c21) in cultured rat brain astrocytes by a combination of mass spectroscopy and molecular biology techniques. This is a significant finding as expression of 21-hydroxylase within astrocytes may be indicative of a wider role for these cells in modulating neuronal behaviour.

Spawning season, fecundity and proximate composition of the gonads of wild-caught blacklip abalone (Haliotis rubra) from Port Fairy waters, south eastern Australia

This study was based on wild-caught blacklip abalone Haliotis rubra L., from Port Fairy waters, south eastern Australia (142°15′E; 38°21′S), from July  1998 to November 1999, and was initiated to evaluate the spawning season and other aspects of its reproductive biology. The shell length and body weight of female and male abalone sampled ranged from 12.0 to 18.6 and 12 to 15 cm, and 137 to 529 and 148 to 585 g, respectively. The sex ratio did not vary significantly from 1:1 through the year. The gonadosomatic index (GSI) ranged from 3.0% to 8.4% in males, and 2.5% to 14.1% for females, and the highest GSI as well as the highest proportion of mature animals were recorded from September to October. During these months the hepatosomatic index (HSI) was low, and an inverse correlation between GSI and HSI (P < 0.05) was evident. Fecundity of blacklip abalone ranged from 1.09 to 7.5 million eggs for females of 12–14.5 cm in length, and 115–487 g in total body weight, respectively. The lipid content of the female gonad increased significantly from about July to November, and an opposite trend was observed for lipid content of the digestive gland. Seasonal changes in the protein and ash contents of the gonad and/or the digestive gland were not always significant.

Zinc deficiency and its inherited disorders - A review

Zinc is an essential trace element required by all living organisms because of its critical roles both as a structural component of proteins and as a cofactor in enzyme catalysis. The importance of zinc in human metabolism is illustrated by the effects of zinc deficiency, which include a diminished immune response, reduced healing and neurological disorders. Furthermore, nutritional zinc deficiency can be fatal in newborn or growing animals. While zinc deficiency is commonly caused by dietary factors, several inherited defects of zinc deficiency have been identified. Acrodermatitis enteropathica is the most commonly described inherited condition found in humans. In several of the few cases that have been reported, this disorder is associated with mutations in the hZIP4 gene, a member of the SLC39 family, whose members encode membranebound putative zinc transporters. Mutations in other members of this family or in different genes may account for other cases of acrodermatitis in which defects in hZIP4 have not been detected. Another inherited form of zinc deficiency occurs in the lethal milk mouse, where a mutation in ZnT4 gene, a member of the SLC30 family of transmembrane proteins results in impaired secretion of zinc into milk from the mammary gland. A similar disorder to the lethal milk mouse occurs in humans. In the few cases studied, no changes in ZnT4 orthologue, hZnT4, were detected. This, and the presence of several minor phenotypic differences between the zinc deficiency in humans and mice, suggests that the human condition is caused by defects in genes that are yet to be identified. Taking into account the fact that there are no definitive tests for zinc deficiency and that this disorder can go undiagnosed, plus the recent identification of multiple members of the SCL30 and SLC39, it is likely that mutations in other genes may underlie additional inherited disorders of zinc deficiency.

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

The execution of Van Nguyen: an Australian perspective

The execution of 25-year-old Melbourne man, Van Nguyen, by Singaporean authorities on 2 December 2005 for attempting to smuggle 400 grams of heroin out of Singapore was cruel. It was also futile. Yet, there are three important lessons that can be learned from his killing. The first lesson is that if Australia is to exert genuine moral pressure on nations to abolish the death penalty it must do so in a principled manner, rather than making expedient pleas when Australians happen to be on the wrong end of the cruel practice. Secondly, sentencing practice in Australia, while not condoning capital punishment, is unjustifiably punitive and we should ameliorate the harshness of some of our sentencing laws. Finally, the death of Nguyen, while tragic, was no more tragic than the millions of other preventable deaths that occur daily throughout the world. The compassion displayed toward Nguyen should be used as a catalyst for enlarging our sympathy gland in relation to all preventable deaths. I now discuss these in further detail.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Extremely high conservation in the untranslated Region as well as the coding region of CNP mRNAs throughout elasmobranch species

C-type natriuretic peptide (CNP) is a crucial osmoregulatory hormone in elasmobranchs, participating in salt secretion and drinking. In contrast to teleosts and tetrapods in which the NP family is composed of a group of structurally related peptides, we have shown that CNP is the sole NP in sharks. In the present study, CNP cDNAs were cloned from four species of batoids, another group of elasmobranchs. The cloned batoid CNP precursors contained a plausible mature peptide of 22 amino acid residues that is identical to most shark CNP-22s, but five successive amino acids were consistently deleted in the prosegment compared with shark precursors, supporting the diphyletic classification of sharks and rays. In addition, molecular phylogenetic trees of CNP precursors were consistent with a diphyletic interpretation. Except for the deletion, the nucleotide and deduced amino acid sequences of the CNP cDNAs are extremely well-conserved among all elasmobranch species, even between sharks and rays. Surprisingly, high conservation is evident not only for the coding region, but also for the untranslated regions. It is most likely that the high conservation is due to the low nucleotide substitution rate in the elasmobranch genome, and high selection pressure. The 3′-untranslated region of the elasmobranch CNP cDNAs contained three to six repeats of the ATTTA motif that is associated with the regulation of mRNA stability and translation efficiency. Alternative polyadenylation sites were also found; the long 3′-untranslated region contains a core of ATTTA motifs while the short form has only one or no ATTTA motif, indicating that the post-transcriptional modification of mRNA is important for regulation of CNP synthesis. These characteristics in the 3′-untranslated region were conserved among all elasmobranch CNP cDNAs. Since CNP has been implicated as a fast-acting hormone to facilitate salt secretion from the rectal gland, the conserved 3′-untranslated region most likely contributes to rapid regulation of CNP synthesis in elasmobranchs in response to acute changes in internal and external environments.

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background
Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.


Methods
We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.


Results
We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.


Conclusion
We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

The lactation cycle of the fur seal

The fur seal is a mammal with an unusual ability to turn its milk production on and off without significantly altering the gross morphology of the mammary gland. This atypical lactation cycle is due to the fact that maternal foraging and infant nursing are spatially and temporally separate (Bonner, 1984). Maternal care involves the suckling of offspring over a period of at least 4 months, but lactation can extend to more than 12 months. Following a perinatal fast of approximately 1 week, females depart the breeding colony to forage at sea and, for the remainder of lactation, alternate between short periods ashore suckling their young with longer periods of up to 4 weeks foraging at sea. Whilst foraging at sea, milk production in the fur seal mammary gland either ceases or is reduced (Arnould & Boyd, 1995b).

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Effects of melatonin on hemolymph glucose and lactate concentrations in the fiddler crab, uca pugilator

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine hormone produced by the pineal gland that works to regulate sleep/wake cycles and activity rhythms. The effects of melatonin in metabolism are far from understood. Melatonin was injected into the fiddler crab, Uca pugilator, to investigate the effects of melatonin on hemolymph glucose and lactate levels. Following injection at t=O, hemolymph samples were collected at t=O.5, 1.0, 1.5 and 5.0 hours. Melatonin caused a decrease in the stress response to injection and also caused delayed hyperglycemia. Melatonin-injected crabs also retained the glucose and lactate rhythymicity when compared to saline-injected crabs. Glucose and lactate rhythms followed the same pattern indicating that the cycles are coupled. Also, melatonin was synthesized using tbe Fischer Indole synthesis and characterized using H?NMR. The synthetic melatonin demonstrated biological activity when injected into the crabs as when compared to pure melatonin on the effects on glucose and lactate concentrations. Overall, melatonin influences both glucose metabolism and the production of lactate.

Osmoregulation in elephant fish, CALLORHYNCHUS MILLII (HOLOCEPHALI)

Osmoregulatory mechanisms in holocephalan fishes are unknown except that they conduct urea-based osmoregulation as in elasmobranchs. We, therefore, examined changes in plasma parameters of elephant fish, Callorhynchus millii, after gradual transfer to concentrated (120%) or diluted (80%) seawater (SW). In control fish, plasma Na and urea concentrations were about 300mM and 450mM, respectively. These values were equivalent to those of sharks and rays, but the plasma urea concentration of elephant fish was considerably higher than that reported for chimaeras, another holocephalan. After transfer to 120% SW, the plasma Na concentration markedly increased, while a conspicuous decrease in plasmaurea concentration was observed following transfer to 80% SW. In elephant fish, we could not find a discrete rectal gland. Instead, approximately 10 tubular structures were located in the wall of post-valvular intestine. Each tubular structure was composed of a putative salt-secreting component consisting of a single-layered columnar epithelium, which was stained with anti-Na+,K±ATPase serum. It is most likely that the tubular structures in the posterior intestine represent a primitive form of the rectal gland in elephant fish. In addition, we have identified two C-type natriuretic peptides (CNPs) from the heart and brain of elephant fish, which may contribute to the control of NaCl excretion from the rectal gland of elephant fish as it does in elasmobranchs.