Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

The binding of androgen receptor to DNA and RNA

The androgen receptor from mouse kidney cytosol has been studied for its nucleic acid binding properties by DNA-cellulose centrifugation assay. The receptor appears to bind to RNA (mRNA, tRNA, rRNA) as well as to DNA. Salt and heat activation of the androgen receptor enhances both DNA and RNA binding. The receptor binds slightly better to denatured DNA than to native DNA. The androgen receptor binds about 2-fold tighter to poly(dG-dC) than to poly (dA-dT). The interaction of the receptor with DNA is not greatly affected by the BrdUrd substitution. The observation that androgen receptor shows a significant affinity to RNA may imply that androgen receptor-RNA interaction could play a role in gene regulation.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

The carbamate reaction of glycylglycine, plasma, and tissue extracts evaluated by a pH stopped flow apparatus.

We have used a stopped flow rapid reaction pH apparatus to investigate the carbamate equilibrium in glycylglycine solutions and in three biological tissues, human plasma, sheep muscle, and sheep brain, as well as to investigate the kinetics of carbamate formation in glyclyglycine solution and in human plasma. The rapid reaction apparatus was equipped with a pH sensitive glass electrode in order to follow the time course of pH from 0.005 to 100 s after rapid mixing of a solution of amine or protein and CO2. Two phases of the pH curve were observed: a fast phase representing carbamate formation, and a slow phase due to the hydration of CO2 which was uncatalyzed since a carbonic anhydrase inhibitor was added to the biological solutions. From the time course of pH change during the fast phase K2, the R-NH2 ionization constant, and Kc, the carbamate equilibrium constant as well as the velocity constant for the formation of carbamate, ka could be calculated from data at different pH and pCO2. The carbamate formed in glycylglycine solutions over a wide range of pH and pCO2 was found consistent with the theory of carbamate formation and with published data. At ionic strength 0.16 and 37 degrees pK is 7.67. pKc 4.58. The heat of the carbamate reaction (deltaH) was calculated to be -3.2 kcal/mol between 20 degrees and 37 degrees. Kt of glycylglycine depends quantitatively on ionic strength as predicted by the Debye-Huckel theory. With ionic strength 0.16 ku was found to be 2,500 M1 S1 at 37 degrees. The activation energy of carbamate formation is 6.7 kcal/mol. Carbamate measurements in human plasma at pCO2 from 38 to 359 Torr. pH from 6.9 to 8.3, temperature 37 degrees, and ionic strength 0.15 provided evidence that two kinds of amino groups participate in carbamate formation. From the equilibrium constants computed for the two species they could be identified as alpha- and epsilon-amino groups. On the basis of a protein molecular weight of 69.000. 0.6 alpha-amino groups/molecule with pKz=7.0 and pKc=4.2, and 5.9 epsilon-amino groups/molecule with pKz=9.0 and pKc=4.3 contribute to carbamate formation. The velocity constant ka was estimated to be 4,950 M1 S1 for the alpha-amino groups and 13,800 M1 S1 for the epsilon-amino groups. Under physiological conditions (pCO2=40 Torr. pH=7.4). The concentration of carbamate in plasma is 0.6 mM and the half-time of carbamate formation is 0.05 s. In extracts prepared from sheep brain at 37 degrees pH=7 and pCO2=35 Torr. the carbamate formation was estimated to be 0.8 mM. With pCO2=70 Torr and the same pH and temperature the carbamate concentration in muscle approximates 0.3 mM and increases to 7 mM as pH rises to 8. It is concluded that, as in plasma, a considerable number of epsilon-amino groups appear to be available for carbamate formation in these tissues.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.     

Sensitivity of the S1 neuronal calcium network to insulin and Bay‐K 8644 in vivo: Relationship to gait,motivation, and aging processes

Neuronal hippocampal Ca²⁺ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer''s disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca²⁺ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca²⁺ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single‐cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging‐ and Ca²⁺‐dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L‐type voltage‐gated Ca²⁺ channel modifier Bay‐K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca²⁺ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age.