Effects of pancreas cold ischemia on islet function and quality.

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.

Long-term changes in glutamatergic synaptic transmission in phenylketonuria.

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.

Nucleotide sequence and evolution of the orangutan epsilon globin gene region and surrounding Alu repeats.

We have mapped and sequenced the epsilon globin gene and seven surrounding Alu repeat sequences in the orangutan beta globin gene cluster and have compared these and other orangutan sequences to orthologously related human sequences. Noncoding flanking and intron sequences, synonymous sites of alpha, gamma, and epsilon globin coding regions, and Alu sequences in human and orangutan diverge by 3.2%, 2.7%, and 3.7%, respectively. These values compare to 3.6% from DNA hybridizations and 3.4% from the psi eta globin gene region. If as suggested by fossil evidence and "molecular clock" calculations, human and orangutan lineages diverged about 10-15 MYA, the rate of noncoding DNA evolution in the two species is 1.0-1.5 X 10(-9) substitutions per site per year. We found no evidence for either the addition or deletion of Alu sequences from the beta globin gene cluster nor is there any evidence for recent concerted evolution among the Alu sequences examined. Both phylogenetic and phenetic distance analyses suggest that Alu sequences within the alpha and beta globin gene clusters arose close to the time of simian and prosimian primate divergence (about 50-60 MYA). We conclude that Alu sequences have been evolving at the rate typical of noncoding DNA for the majority of primate history.