Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

What is Microthrix parvicella ?

Microthrix parvicella is one of the filamentous bacteria that is known to create problems in the operation of activated sludge plants. Its physiology has already been investigated, although primarily in the context of its being a bulking species. It is now recognized that it is one of the major foam-forming organisms and, as such, needs further study. The initial isolation of M. parvicella did not prove to be as easy as would be expected from the earlier work and, eventually, micromanipulation was required. Growth studies showed that it exhibited several morphological forms, only one of which was that described previously. This has led to doubts about the classification of this species which does not yet have a clearly defined bacteriological designation.     

Recent advances in JAK3 inhibition: Isoform selectivity by covalent cysteine targeting

Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clinical validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compounds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compounds demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clinical studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clinical efficacy.     

A Low Cost Metal-Free Vascular Access Mini-Port for Artifact Free Imaging and Repeated Injections in Mice

Purpose

Small injection ports for mice are increasingly used for drug testing or when administering contrast agents. Commercially available mini-ports are expensive single-use items that cause imaging-artifacts. We developed and tested an artifact-free, low-cost, vascular access mini-port (VAMP) for mice.

Procedures

Leakage testing of the VAMP was conducted with high speed bolus injections of different contrast agents. VAMP-induced artifacts were assessed using a micro-CT and a small animal MRI (9.4T) scanner ex vivo. Repeated contrast administration was performed in vivo.

Results

With the VAMP there was no evidence of leakage with repeated punctures, high speed bolus contrast injections, and drawing of blood samples. In contrast to the tested commercially available ports, the VAMP did not cause artifacts with MRI or CT imaging.

Conclusions

The VAMP is an alternative to commercially available mini-ports and has useful applications in animal research involving imaging procedures and contrast agent testing.     

Urotensin II and urotensin II-related peptide (URP) in cardiac ischemia-reperfusion injury

Circulating urotensin II (UII) concentrations and the tissue expression of its cognate receptor (UT) are elevated in patients with cardiovascular disease (CVD). The functional significance of elevated plasma UII levels in CVD is unclear. Urotensin-related peptide (URP) is a paralog of UII in that it contains the six amino acid ring structures found in UII. Although both peptides are implicated as bioactive factors capable of modulating cardiovascular status, the role of both UII and URP in ischemic injury is unknown. Accordingly, we provide here the first report describing the direct cardiac effects of UII and URP in ischemia-reperfusion injury. Isolated perfused rat hearts were subjected to no-flow global ischemia for 45 min after 30min preconditioning with either 1nM rUII or 10nM URP. Both rUII- and URP-induced significant vasodilation of coronary arteries before (both P<0.05) and after ischemia (both P<0.05). Rat UII alone lowered contractility prior to ischemia (P=0.053). Specific assay of perfusate revealed rUII and URP both significantly inhibited reperfusion myocardial creatine kinase (CK) release (P=0.012 and 0.036, respectively) and atrial natriuretic peptide (ANP) secretion (P=0.025). Antagonism of the UT receptor with 1muM palosuran caused a significant increase in perfusion pressure (PP) prior to and post-ischemia. Furthermore, palosuran significantly inhibited reductions in both PP and myocardial damage marker release induced by both rUII and URP. In conclusion, our data suggests rUII and URP reduce cardiac ischemia-reperfusion injury by increasing flow through the coronary circulation, reducing contractility and therefore myocardial energy demand, and inhibiting reperfusion myocardial damage. Thus, UII and URP present as novel peptides with potential cardioprotective actions.     

Structural Fold and Binding Sites of the Human Na-Phosphate Cotransporter NaPi-II

Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in P_i homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na⁺-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na⁺ and P_i coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.     

Perturbations in three medullary nuclei enhance fractionated breathing in awake goats.

Our aim was to determine the frequency and characteristics of a fractionated pattern of diaphragm and upper airway muscle activity and airflow during wakefulness and sleep in adult goats. A fractionated breath (FBr) was defined as three or more brief (40-150 ms) interruptions in the diaphragm activity not associated with multiple swallows, eructation, mastication, or movement. During a FBr, the discharge pattern in the diaphragm and upper airway muscles showed complete cycles of inspiration and expiration. Whereas the interval between peak diaphragm activity of the breath preceding the FBr to the first diaphragm peak of the FBr was 15-20% less than the average interval of the preceding five control breaths, the breath-to-breath interval of the five breaths after a FBr did not differ from the control breaths before the FBr event. In normal goats, FBr was evident in only 4 of 18 (22%) awake goats and in only one of these goats during non-rapid eye movement sleep. In 35 goats with implanted microtubules in the medulla, FBr were present in 14 (40%) goats. In these goats with FBr, 78% (11 of 14) had one or more implantations into or near the facial, vestibular, or raphe nuclei. The effect of perturbations in these nuclei is probably nonspecific, because injections into these nuclei with mock cerebrospinal fluid or excitatory amino acid-receptor agonist or antagonist produced both increases and decreases in the frequency of the FBr while not altering their characteristics. Finally, a swallow occurred at the termination or during the first breath after 60% of the FBr. We speculate that the FBr manifest 1) the disruption of a neuronal network, which coordinates breathing and other functions (such as swallowing), utilizing the same anatomic structures, and/or 2) transient changes in synaptic inputs that increase the rate of the normal respiratory rhythm generator or allow an ectopic, anomalous generator to become dominant.     

Cost-effectiveness of pharmacotherapy to reduce obesity

Aims

Obesity causes a high disease burden in Australia and across the world. We aimed to analyse the cost-effectiveness of weight reduction with pharmacotherapy in Australia, and to assess its potential to reduce the disease burden due to excess body weight.

Methods

We constructed a multi-state life-table based Markov model in Excel in which body weight influences the incidence of stroke, ischemic heart disease, hypertensive heart disease, diabetes mellitus, osteoarthritis, post-menopausal breast cancer, colon cancer, endometrial cancer and kidney cancer. We use data on effectiveness identified from PubMed searches, on mortality from Australian Bureau of Statistics, on disease costs from the Australian Institute of Health and Welfare, and on drug costs from the Department of Health and Ageing. We evaluate 1-year pharmacological interventions with sibutramine and orlistat targeting obese Australian adults free of obesity-related disease. We use a lifetime horizon for costs and health outcomes and a health sector perspective for costs. Incremental Cost-Effectiveness Ratios (ICERs) below A$50 000 per Disability Adjusted Life Year (DALY) averted are considered good value for money.

Results

The ICERs are A$130 000/DALY (95% uncertainty interval [UI] 93 000–180 000) for sibutramine and A$230 000/DALY (170 000–340 000) for orlistat. The interventions reduce the body weight-related disease burden at the population level by 0.2% and 0.1%, respectively. Modest weight loss during the interventions, rapid post-intervention weight regain and low adherence limit the health benefits.

Conclusions

Treatment with sibutramine or orlistat is not cost-effective from an Australian health sector perspective and has a negligible impact on the total body weight-related disease burden.