Effects of germination on the activities of amylases and phenolic enzymes in sorghum varieties grouped according to food end‐use properties

Fifty sorghum varieties were screened to determine the effects of germination on levels of starch, α‐amylase, β‐amylase, phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO). Germination decreased starch content, with amylose being more degraded than amylopectin. In germinated grain, α‐amylase activity increased several‐fold in all varieties, whereas β‐amylase activity did not increase uniformly and even decreased in some varieties. Activity of the key enzyme in phenolic biosynthesis, PAL, was detected in only half of the varieties before germination but in all of them after germination. PPO was not activated in germinated sorghum grains, whereas POX activity increased up to tenfold in some varieties. Zymography revealed that germination induced de novo synthesis of several POX isoenzymes, among which an anionic POX isoenzyme (pI 3.1) was ubiquitously present. Amylase and phenolic enzyme activities could be correlated with grain and plant agronomic characteristics. The use of sorghum varieties for local dishes such as ‘tô’, ‘dolo’, couscous and thin porridge could be correlated with amylase and phenolic enzyme activities and the contents of their substrates. The biochemical constituents determined are useful markers for selection of varieties for food utilisation with special emphasis on infant porridges. Copyright © 2006 Society of Chemical Industry     

Effects of central and peripheral administration of kynurenic acid on hippocampal evoked responses in vivo and in vitro

Kynurenic acid is an excitatory amino acid antagonist with preferential activity at the N-methyl-D-aspartate subtype of glutamate receptors. It is produced endogenously in the brain, but is synthesized more effectively in the periphery. The influence of peripheral kynurenic acid on brain function is unclear because kynurenic acid is likely to penetrate the blood-brain barrier poorly. To determine the potential central effects of peripheral kynurenic acid, we compared its effects in the hippocampus after peripheral or direct administration. The hippocampus of the rat was chosen as a test system because this region receives glutamatergic inputs, and because responses to stimulation of these inputs can be compared after peripheral drug administration in vivo, and after direct administration of drugs in vitro. Peripherally-administered kynurenic acid was injected via a catheter in the jugular vein. Bath-application to hippocampal slices was used to test effects of direct administration. Area CA1 pyramidal cells and dentate gyrus granule cells were examined by extracellular recording and stimulation of area CA3 or the perforant path, respectively. Pairs of identical stimuli were used to assess paired-pulse inhibition and paired-pulse facilitation. Kynurenic acid decreased evoked responses in area CA1 and the dentate gyrus, both in vivo and in vitro. Effective concentrations were in the low micromolar range, and therefore were likely to be mediated by antagonism of N-methyl-D-aspartate receptors. In both preparations, area CA1 was more sensitive than the dentate gyrus, and paired-pulse facilitation was affected, but not paired-pulse inhibition. Control solutions had no effect. We conclude that kynurenic acid can enter the brain after peripheral administration, and that peripheral and direct effects in the hippocampus are qualitatively similar. Therefore, we predict that effects of endogenous kynurenic acid that was synthesized peripherally or centrally would be similar. Furthermore, the results suggest that modulation of the glycine site of the N-methyl-D-aspartate receptor, for example by kynurenic acid, may vary considerably among different brain areas.     

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.     

Contribution of plasminogen activators and their inhibitors to the survival prognosis of patients with Dukes' stage B and C colorectal cancer

Despite the advances in pre-, peri- and post-operative medical care of colorectal carcinoma patients, the prognosis has improved only marginally over recent decades. Thus, additional prognostic indicators would be of great clinical value to select patients for adjuvant therapy. In previous studies we found that colorectal carcinomas have a marked increase of the urokinase-type of plasminogen activator (u-PA), and the inhibitors PAI-1 and PAI-2, whereas the tissue-type plasminogen activator (t-PA) is found to be decreased in comparison with adjacent normal mucosa. In the present study we evaluated the prognostic value of several plasminogen activation parameters, determined in both normal and carcinomatous tissue from colorectal resection specimens, for overall survival of 136 Dukes' stage B and C colorectal cancer patients, in relation to major clinicopathological parameters. Uni- and multivariate analyses indicated that a high PAI-2 antigen level in carcinoma, a low t-PA activity and antigen level and a high u-PA/t-PA antigen ratio in adjacent normal mucosa are significantly associated with a poor overall survival. A high ratio of u-PA antigen in the carcinomas and t-PA antigen in normal mucosa, i.e. u-PA(C)/t-PA(N), was found to be predictive of a poor overall survival as well. All these parameters were found to be prognostically independent of the clinicopathological parameters. Multivariate analysis of combinations of these prognostically significant plasminogen activation parameters revealed that they are important independent prognostic indicators and have in fact a better prognostic value than their separate components. Based on these combined parameters, subgroups of patients with Dukes' stage B and C colorectal cancer could be identified as having either a high or a low risk regarding overall survival. In conclusion, these findings emphasize the relevance of the intestinal plasminogen activation system for survival prognosis of patients with colorectal cancer and, in the future, might constitute a patient selection criterion for adjuvant therapy.     

Temporal aspects of the effects of cooling on responses of single auditory nerve fibers

During an investigation of the effects of cochlear cooling on frequency tuning and input/output relations of single auditory nerve fibers in gerbil (Ohlemiller and Siegel (1994) Hear. Res. 80, 174-190), cooling-related changes in post-stimulus time histogram (PSTH) shape and phase-locking to tonebursts were characterized in a small sample of neurons. Local cochlear cooling by 5-10 degrees C below normal core temperature did not alter overall PSTH shape, although some evidence was found for a reduction in the time constants of rapid and short term rate adaptation. The relative contributions of rapid and short term response components appeared unaltered. Effects of cooling on phase-locking were assessed by calculating the synchronization index for responses to intense ( > 70 dB SPL) tonebursts at 0.5, 1.0, and 2.0 kHz. Synchronization filter functions exhibited modest reductions in both magnitude and the upper frequency limit of phase-locking. The effects of cooling on the temporal character of responses appear distinct from those of a simple reduction in stimulus intensity. Results are interpreted in terms of cooling-related changes in responses of cochlear hair cells and afferent neurons, and suggest that temperature artifacts are unlikely to underlie reported species differences in PSTH shape and phase-locking.