Detection of calmodulin-binding proteins and calmodulin-dependent phosphorylation linked to calmodulin-dependent chemotaxis to folic and cAMP in Dictyostelium

Calmodulin (CaM) antagonists, trifluoperazine (TFP) or calmidazolium (R24571), dose-dependently inhibited cAMP and folic acid (FA) chemotaxis in Dictyostelium. Developing, starved, and refed cells were compared to determine if certain CaM-binding proteins (CaMBPs) and CaM-dependent phosphorylation events could be identified as potential downstream effectors. Recombinant CaM ([35S]VU-1-CaM) gel overlays coupled with cell fractionation revealed at least three dozen Ca(2+)-dependent and around 12 Ca(2+)-independent CaMBPs in Dictyostelium. The CaMBPs associated with early development were also found in experimentally starved cells (cAMP chemotaxis), but were different for the CaMBP population linked to growth-phase cells (FA chemotaxis). Probing Western blots with phosphoserine antibodies revealed several phosphoprotein bands that displayed increases when cAMP-responsive cells were treated with TFP. In FA-responsive cells, several but distinct phosphoproteins decreased when treated with TFP. These data show that unique CaMBPs are present in growing, FA-chemosensitive cells vs. starved cAMP-chemoresponsive cells that may be important for mediating CaM-dependent events during chemotaxis.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Functional characteristics of dystrophic skeletal muscle: insights from animal models.

Muscular dystrophies are a clinically and genetically heterogeneous group of disorders that show myofiber degeneration and regeneration. Identification of animal models of muscular dystrophy has been instrumental in research on the pathogenesis, pathophysiology, and treatment of these disorders. We review our understanding of the functional status of dystrophic skeletal muscle from selected animal models with a focus on 1) the mdx mouse model of Duchenne muscular dystrophy, 2) the Bio 14.6 delta-sarcoglycan-deficient hamster model of limb-girdle muscular dystrophy, and 3) transgenic null mutant murine lines of sarcoglycan (alpha, beta, delta, and gamma) deficiencies. Although biochemical data from these models suggest that the dystrophin-sarcoglycan-dystroglycan-laminin network is critical for structural integrity of the myofiber plasma membrane, emerging studies of muscle physiology suggest a more complex picture, with specific functional deficits varying considerably from muscle to muscle and model to model. It is likely that changes in muscle structure and function, downstream of the specific, primary biochemical deficiency, may alter muscle contractile properties.     

Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Loss of calcineurin from the medial preoptic area of primiparous rats

Western blot analyses reveal that calcineurin A (CNA), which is present in the hippocampus, basolateral amygdala, parietal cortex, and MPOA of virgin males and females, is undetectable only in the MPOA of primiparous females regardless of whether they had postpartum pup contact or not. In contrast, CNB was expressed at unchanging levels in the PC and MPOA. Similarly, G(alphao) and PKA(RI) were expressed at high levels in all of the brain regions of virgin males, virgin females, and primiparous females, supporting the concept that this loss of CNA is a specific event. Understanding how and why the expression of CNA, the sole neuronal Ca2+/CaM-dependent protein phosphatase, is down-regulated specifically in the MPOA of primiparous females may yield some insight into the signal transduction events that mediate the onset of mammalian maternal behavior.     

Congenital fibrosis of the vertically acting extraocular muscles maps to the <Emphasis Type="Italic">FEOM3</Emphasis> locus

The diagnosis of congenital fibrosis of the extraocular muscles (CFEOM) encompasses several different inherited strabismus syndromes characterized by congenital restrictive ophthalmoplegia affecting extraocular muscles innervated by the oculomotor and/or trochlear nerves. The OMIM database (http://www.ncbi.nlm.nih.gov/Omim/) currently contains four familial CFEOM phenotypes: CFEOM1-3, which map to the FEOM1-3 loci (MIM 135600, 602078, 604361), respectively, and congenital fibrosis of the vertically acting extraocular muscles (MIM 600638), reported in a single family without a corresponding genotype. We have had the opportunity to study the reported family with this fourth phenotype and now demonstrate that their phenotype can be reclassified as CFEOM3 and that it maps to FEOM3, flanked by D16S498 to 16qter, with a maximum lod score of 6.0.