Enhancement of Pig Embryonic Implants in Factor VIII KO Mice: A Novel Role for the Coagulation Cascade in Organ Size Control

Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.     

Backbone cyclic pheromone biosynthesis activating neuropeptide (PBAN) antagonists: inhibition of melanization in the moth Spodoptera littoralis (Insecta, Lepidoptera)

Antagonistic and agonistic activities of backbone cyclic (BBC) pheromone biosynthesis activating neuropeptide (PBAN) analogues were evaluated in an attempt to identify potent melanotropic antagonists, to gain an insight into their structure-activity relationship (SAR), and to discover molecules with selective and non-selective melanotropic and pheromonotropic properties. Eight potent melanotropic BBC antagonists and seven agonists were disclosed. SAR studies revealed that the structural requirements of the melanotropic and pheromonotropic agonists and antagonists are different. The cyclic structure of the BBC peptides was unimportant for antagonistic activity, and linearization retained their melanotropic and pheromonotropic antagonistic properties. Comparison of the antagonistic activities of the BBC and precyclic peptides with respect to both functions revealed eight selective antagonists (six that were selective melanotropic antagonists and two selective pheromonotropic antagonists) and four non-selective (melanotropic and pheromonotropic) antagonists. The selective melanotropic antagonists exhibited both, pure or mixed agonistic/antagonistic activities. The selective pheromonotropic compounds were pure antagonists. All non-selective compounds were pure antagonists. Comparison of the agonistic activities of the BBC peptides with respect to both functions revealed six selective melanotropic agonists and one non-selective agonistic compound. All compounds (whether selective or non-selective) exhibited pure agonistic activity. Discovery of the selective compounds hints at the possibility that the receptors that mediate the respective activities may have different properties.     

Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

Summary In the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10^–11–10^–7 m) was found to stimulate²²Na^– uptake by the isolated BBM vesicles directly. AII did not affect the Na⁺-dependent BBM glucose uptake, and the effect of AII on BBM²²Na⁺ uptake was inhibited by amiloride, suggesting the involvement of Na⁺/H⁺ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na⁺/H⁺ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A₂ (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and²²Na⁺ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM²²Na⁺ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM²²Na⁺ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM²²Na⁺ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na⁺/H⁺ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.     

Helicobacter pylori: the Middle East scenario.

A review of Helicobacter pylori in the Middle East is presented. Prevalence studies have been performed in asymptomatic population groups from Algeria, Israel, Saudi Arabia and Turkey. These showed that the prevalence of H. pylori is similar to that of the developing countries of the world with a high level of infection in childhood (40 to 70 percent), which increases with age to 85 to 90 percent. Israel, however, has a low prevalence in children (10 percent), but there is a rapid rise in the second decade of life to 39 percent, reaching 79 percent in those over 60 years old. The prevalence rates were higher in those living in communal settlements (72 percent) than in urban dwellers (65 percent). The infection rates were higher in persons of Mediterranean and Asian origin (89 percent) compared to those of Western European/North American origin (57 percent). The prevalence rate of H. pylori infection in patients undergoing endoscopy for upper gastrointestinal symptoms has now been reported from many Middle Eastern countries, including Egypt, Iran, Israel, Oman, Saudi Arabia, the United Arab Emirates and Yemen. These studies showed that patients with gastritis and peptic ulcer disease had similar rates of infection as reported from Europe, United States and Africa (71 to 92 percent). However, patients with non-ulcer dyspepsia had higher rates of infection (61 to 89 percent). The H. pylori scenario from the prevalence rates, treatment protocols and responses to treatment does not differ very much from other developing areas of the world.     

The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.     

Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.     

Development of bifunctional photoactivatable benzophenone probes and their application to glycoside substrates

Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin.     

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold-adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered "contradictory" to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin-like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP-dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation.