Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection

Iron acquisition, mediated by specific outer membrane receptors, is critical for colonization of the urinary tract by uropathogenic Escherichia coli (UPEC). The role of specific iron sources in vivo , however, remains largely unknown. In this study, we identified a 79 kDa haem receptor, h ae m a cquisition protein Hma, and established that it functions independently of ChuA to mediate haemin uptake by UPEC strain CFT073. We demonstrated that expression of hma promotes TonB-dependent haemin utilization and the Hma protein binds haemin with high affinity ( K _d = 8 μM). Hma, however, lacks conserved His residues shown to mediate haem uptake by other bacterial receptors. In contrast, we identified Tyr-126 as a residue necessary for Hma-mediated haemin utilization. In a murine co-infection model of UTI, an isogenic hma mutant was out-competed by wild-type CFT073 in the kidneys ( P  < 0.001) and spleens ( P  <  0.0001) of infected mice, indicating its expression provided a competitive advantage in these organs. Furthermore, a hma chuA double mutant, which is unable to utilize haemin, was unable to colonize the kidneys to wild-type levels during independent infection ( P  = 0.02). Thus, we demonstrate that UPEC requires haem for kidney colonization and that uptake of this iron source is mediated, in part, by the novel receptor, Hma.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Initiation of Swarming Motility by Proteus mirabilis Occurs in Response to Specific Cues Present in Urine and Requires Excess l-Glutamine

Proteus mirabilis, a leading cause of catheter-associated urinary tract infection (CaUTI), differentiates into swarm cells that migrate across catheter surfaces and medium solidified with 1.5% agar. While many genes and nutrient requirements involved in the swarming process have been identified, few studies have addressed the signals that promote initiation of swarming following initial contact with a surface. In this study, we show that P. mirabilis CaUTI isolates initiate swarming in response to specific nutrients and environmental cues. Thirty-three compounds, including amino acids, polyamines, fatty acids, and tricarboxylic acid (TCA) cycle intermediates, were tested for the ability to promote swarming when added to normally nonpermissive media. l-Arginine, l-glutamine, dl-histidine, malate, and dl-ornithine promoted swarming on several types of media without enhancing swimming motility or growth rate. Testing of isogenic mutants revealed that swarming in response to the cues required putrescine biosynthesis and pathways involved in amino acid metabolism. Furthermore, excess glutamine was found to be a strict requirement for swarming on normal swarm agar in addition to being a swarming cue under normally nonpermissive conditions. We thus conclude that initiation of swarming occurs in response to specific cues and that manipulating concentrations of key nutrient cues can signal whether or not a particular environment is permissive for swarming.     

Characterization of FKGK18 as Inhibitor of Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β): Candidate Drug for Preventing Beta-Cell Apoptosis and Diabetes

Ongoing studies suggest an important role for iPLA₂β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA₂βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA₂β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA₂ than of cPLA₂ or sPLA₂, leading to its designation as a “specific” inhibitor of iPLA₂, it has been shown to also inhibit non-PLA₂ enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA₂β and membrane-associated iPLA₂γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA₂β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA₂β with a greater potency (100-fold) than iPLA₂γ, (b) inhibition of iPLA₂β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA₂β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA₂β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA₂β role in biological functions.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

The Obesity Epidemic and Its Impact on Urologic Care

Although heart disease and cancer are the number one and two causes of death in the United States, respectively, obesity is gaining speed as a contributing cause to both of those conditions, along with diabetes, arthritis, dyslipidemia, coronary heart disease, gallbladder disease, and certain malignancies. Nearly one-third of the adults in the United States is overweight with a body mass index (BMI) greater than 25 kg/m², and another third of the adult population is obese, with a BMI greater than 30 kg/m². This article reviews the root causes of obesity, the societal implications, and the implications of obesity on various urologic diseases.Key words: Obesity, Morbid obesity, Body mass index, Exercise, Weight loss, Diet, EpidemicMore than 20% of adults in the United States are clinically obese, defined by a body mass index (BMI) of 30 kg/m² or higher, and an additional 30% are overweight, with a BMI between 25 and 30 kg/m².¹ An environment that promotes excessive food intake and discourages physical activity lies at the root of the current obesity epidemic. Although humans have excellent physiologic mechanisms to defend against body weight loss, they have only weak physiologic mechanisms to defend against body weight gain when food is abundant. So much has been discussed about the obesity epidemic that it’s easy to think the issue is being blown out of proportion. After all, people putting on a few pounds may not seem to warrant the proclamation of a national emergency. Although obesity may not attract the degree of attention that heart disease and cancer do, it is a serious public health issue. Experts agree that, as more and more obese children become obese adults, the diseases associated with obesity, such as heart disease, cancer, and particularly diabetes, will surge.The obesity epidemic in the United States is an unintended consequence of the economic, social, and technologic advances realized during the past several decades. The food supply is abundant and low in cost, and palatable foods with high caloric density are readily available in prepackaged forms and at fast-food restaurants. Laborsaving technologies have greatly reduced the amount of physical activity that used to be part of everyday life, and the widespread availability of electronic devices in the home, school, and office has promoted a sedentary lifestyle, particularly among children.A recent study estimated that medical expenditures attributed to overweight and obesity accounted for 9.1% of total US medical expenditures in 1998, and might have reached $78.5 billion dollars.² Today, the healthcare costs attributed to obesity are estimated to be $190 billion—nearly 21% of total US healthcare costs.³ Expenditures will continue to rise, particularly due to increases in the prevalence of obesity and the cost of related healthcare.Total healthcare costs attributable to this obesity epidemic are expected to double every decade, reaching $860.7 to $956.9 billion by 2030, accounting for 16% to 18% of total US healthcare costs, or 1 in every 6 dollars spent on healthcare. ⁴ In addition, obesity is likely to result in a decreased life expectancy for our population. Current US generations may have a shorter life expectancy than their parents if this obesity epidemic cannot be controlled.⁵ Based on nationally representative data and the assumptions of a future of increased obesity rates, along with increased healthcare costs, this paints an alarming picture of the future obesity epidemic. Projections show that if the trends continue, in 15 years, 80% of all American adults will be overweight or obese.⁶