Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells

The exposure of anionic phospholipidson the external surface of injured endothelial cells and activatedplatelets is a primary biological signal to initiate blood coagulation.Disease conditions that promote the formation of ectopic thrombi resultin tissue ischemia. Annexins, Ca²⁺-dependentanionic phospholipid binding proteins, are potential therapeutic agentsfor the inhibition of coagulation. We have designed a transgene thattargets secretion of annexin V from cultured thyroid cells under thecontrol of doxycycline. Our results indicate that annexin V in theendoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis,processing, and secretion of thyroglobulin. ER luminal Ca²⁺was moderately increased and can be released by inositol1,4,5-trisphosphate. Our study demonstrates that targeting andsecretion of annexin V through the secretory pathway of mammalian cellsdoes not adversely affect cellular function. Regulated synthesis andrelease of annexin V may exert anticoagulatory and anti-inflammatoryeffects systemically and may prove useful in further developingtherapeutic strategies for conditions including antiphospholipid syndrome.

     

Phospholipid synthesis,diacylglycerol compartmentation,and apoptosis

Apoptosis is a means by which organisms dispose of unwanted cells without inducing an inflammatory response. Alterations in apoptosis is a common process by which cells become cancerous. Paradoxically, many cancer chemotherapeutics preferentially kill cancer cells by inducing apoptosis. Diacylglycerol is a lipid second messenger that regulates cell growth and apoptosis and is produced during signal transduction by hydrolysis of membrane phospholipids. Protein kinase Cs are a family of diacyglycerol responsive enzymes that are recruited to cellular membranes as a consequence of diacylglycerol production where they phosphorylate specific target proteins responsible for regulating cell growth. In this review, we will first summarize our current understanding of the role of specific proteins kinase C isoforms in the induction of cell growth/apoptosis. Subsequently, we will discuss how insights gained in lipid-mediated regulation of protein kinase Cs promotes our understanding of the role specific family members play in regulating cell growth. Finally, other diacylglycerol binding proteins involved in regulating apoptosis will be discussed.     

Peptides that elicit midgut stem cell differentiation isolated from chymotryptic digests of hemolymph from Lymantria dispar pupae

Isolated stem cells of Heliothis virescens, cultured in vitro, were induced to differentiate by Midgut Differentiation Factors 3 and 4. These were peptides identified from a chymotrypsin digest of hemolymph taken from newly pupated Lymantria dispar. Partial purification was obtained by filtration through size exclusion filters. The most active preparation was subsequently subjected to a series of 3 Reverse Phase-HPLC procedures. Partial sequences of the peptides were identified via automated Edman degradation as the nanomers EEVVKNAIA-OH (MDF 3) and ITPTSSLAT-OH (MDF 4). These sequences were commercially synthesized. The synthetic compounds proved active in a dose-dependent manner. Stem cells responded to synthetic MDF 3 and MDF 4 as they did to previously identified peptides MDF 1 and 2, which have quite different amino acid sequences. All of the 4 MDFs administered singly induced statistically similar differentiation responses at 2 x 10(-8), 2 x 10(-9), and 2 x 10(-10) M. However, pairs of the 4 MDFs produced even more differentiation, the same response as one alone, no response, or were inhibitory, dependent on the MDF pair and its concentration. The data suggests complicated receptor interactions.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Histochemical and SEM evaluation of the neuromuscular junctions from alcoholic rats

In the present study morphological changes occurring in the neuromuscular junctions (NMJ) of the extensor digitorum longus (EDL) and soleus muscles from albino rats (Rattus norvegicus) submitted to experimental chronic alcoholism were evaluated. Seventy two male animals aged 4 months and weighing on average 400g were divided into three groups: control, alcoholic and isocaloric. Six rats from each group were anesthetized and sacrificed after 5, 10, 15 and 18 months. The NMJ did not show detectable morphological changes in either muscle after treatment when examined by light microscopy. With respect to the dimensions, statistical analysis demonstrated a tendency to a statistically significant treatment x time interaction for the length of soleus muscle NMJ. The ultrastructural study, however, revealed that the NMJ of the soleus muscle of animals submitted to 18 months of experimental alcoholism presented important morphological alterations. Characteristically, the NMJ of these muscles is located on an elevation on the surface of the muscle fiber, presenting a regular round, oval or elliptical shape and continuous and not very deep synaptic grooves. Approximately 30% of the NMJ of alcoholic rats are irregular in shape, with the sarcolemmal elevations typical of the synapse region being flattened on at least one side, with discontinuous synaptic grooves, and deep and punctiform contacts of the synaptic buds. These data suggest that, although skeletal muscle has a greater natural resistance against the direct or indirect effects of alcohol, some submicroscopic morphological alterations are detectable in the NMJ, especially in muscles with oxidative metabolism (soleus) following long periods of ingestion of alcohol.     

Identification and functional analysis of a naturally occurring E89K mutation in the ABCA1 gene of the WHAM chicken

The Wisconsin hypoalpha mutant (WHAM) chicken has a >90% reduction in plasma HDL due to hypercatabolism by the kidney of lipid-poor apoA-I. The WHAM chickens have a recessive white skin phenotype caused by a single-gene mutation that maps to the chicken Z-chromosome. This corresponds to human 9q31.1, a chromosomal segment that contains the ATP-binding cassette protein-1 (ABCA1) gene, which is mutated in Tangier Disease and familial hypoalphalipoproteinemia. Complete sequencing of the WHAM ABCA1 cDNA identified a missense mutation near the N-terminus of the protein (E89K). The substitution of this evolutionary conserved glutamate residue for lysine in the mouse ABCA1 transporter leads to complete loss of function, resulting principally from defective intracellular trafficking and very little ABCA1 reaching the plasma membrane. The WHAM chicken is a naturally occurring animal model for Tangier Disease.     

Effects of light and nutrients on plankton stoichiometry and biomass in a P-limited lake

A field enclosure experiment was performed over 12 weeks in a P-limited lake to test the hypothesis that light:nutrient balance affects pelagic communities by altering the C:P stoichiometry of seston and by influencing exudation of labile DOC by algae. Three levels of light intensity (ambient, 50% of ambient, 25% of ambient) were cross-classified with three levels of nutrients in a factorial design (n=2). Dissolved nutrient concentrations, seston C concentration and C:P ratios in small (<1 m) and larger (1–85 m) size fractions were monitored, along with chlorophyll a concentration, abundance of bacteria and protozoa, and biomass and P-content of macrozooplankton. Algal exudation of recently-fixed C into the dissolved pool was also measured at the end of the experiment in selected enclosures. Treatments had no effect on seston C concentration but reduction of light intensity significantly decreased whole seston and large (1–85 m) seston C:P ratios. However, the magnitude of these effects was modest and not likely to be ecologically significant. There were no effects of nutrient addition or light×nutrient interaction on seston stoichiometry. Algae tended to release a higher percentage of fixed C as DOC in high light enclosures but this difference was not statistically significant. There were no effects of treatments on the abundance of bacteria or protozoa but nutrient enrichment led to a statistically significant but generally modest increase in macrozooplankton biomass. No effects on zooplankton community composition or P-content were observed. Comparison of effect sizes and treatment variances indicated a high probability of type II error and thus our confidence in failing to reject the null hypothesis in most of the above cases was low. Thus, our data provide support for only some aspects of the light:nutrient hypothesis but more appropriate tests of the hypothesis should involve stronger treatments and/or increased replication in order to be better able to evaluate its validity.     

Protein folding and three-dimensional domain swapping: a strained relationship?

Many proteins function as multimeric assemblies into which the folded individual promoters organize as higher order structures. An oligomerization mechanism that appears to impose the coordination of events during folding and oligomer assembly is three-dimensional domain swapping. Recent studies have focused on revealing the structural basis of domain swapping and a possible role for domain swapping in the regulation of protein aggregation and activity.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.