Rapid and selective uptake, metabolism, and cellular distribution of docosahexaenoic acid among rod and cone photoreceptor cells in the frog retina

The uptake, metabolism, and cellular distribution of 3H-docosahexaenoic acid (3H-22:6) in the frog retina during in vitro incubation were studied. An initial diffuse labeling throughout the retina was detected by autoradiography and was followed by an active steady increase in labeled photoreceptor cells. After 6 hr of incubation, 92% of the label was concentrated in photoreceptor cells. Among these cells, 435-rods (green rods) labeled heavily and showed two to three times higher uptake than the 502-rods (red rods). Cone uptake labeling was the lowest, showing negligible labeling throughout the cytoplasm. However, oil droplets of the 575-cones actively concentrated 22:6. The high uptake of 3H-22:6 by photoreceptor cells was followed by its rapid esterification into phospholipids. After 6 hr of labeling, only 5% of the radioactivity in the retina was free 22:6, whereas 88% was esterified into phospholipids. The remaining 22:6 was distributed equally in triacylglycerols (TAGs) and diacylglycerols. When 3H-22:6 (0.11 microM) of high specific activity was used, early incubation times showed phosphatidylinositol (PI) labeling to be of the same order of magnitude or greater than that of phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Although the amount of endogenous 22:6 esterified into PI accounted for less than 2% of the 22:6 in retinal phospholipids, 27% of 3H-22:6 labeling was recovered in this phospholipid. When 14C-22:6 at a final concentration of 70 microM was used, a different profile of lipid labeling was observed. Forty percent of the labeling remained in the free fatty acid pool, followed by TAG (24%), PC (14%), and PE (12%). PI showed the smallest increase in picomoles of 14C-22:6 incorporated, when compared with 3H-22:6. In conclusion, a selective and differential uptake of 3H-22:6 by photoreceptor cells is coupled to its active utilization for phospholipid biosynthesis, mainly that of PC, PE, and PI. The differential uptake of 3H-22:6 among photoreceptor cells may reflect involvement of this fatty acid in cell-specific functions.     

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats.

This study investigates the effects of phorbol dibutyrate (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity from the cytosolic to the particulate fraction, in aortas and mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The basal distribution of PKC activity between the cytosolic and particulate fractions of SHR and WKY aortas, and mesenteric arteries, was not significantly different. PDB induced a concentration-dependent decrease in cytosolic PKC activity in SHR and WKY aortas. PDB (0.01 microM) decreased cytosolic PKC activity to a greater magnitude in SHR aorta as compared to WKY aorta, while 1.0 microM PDB decreased cytosolic PKC activities to similar magnitudes in SHR and WKY aortas, and mesenteric arteries. These results suggest that the increased sensitivity of SHR vessels to contraction by phorbol esters may be due, at least in part, to the greater sensitivity of PKC in these vessels to phorbol ester activation.     

Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses

We propose through a sequence and structural-pattern analysis that a protein domain of undefined function encoded by the enveloped RNA flavi- and pestivruses is a Ser active-center enzyme related to the cellular trypsin family. A further homology is emphasized with the group of (Cys active-center) viral proteases encoded by nonenveloped RNA viruses of the picorna-, como-, nepo-, and potyvirus classes. Structural modeling of the putative flaviviral protease domain suggests amino acids that are crucial for catalytic activity and substrate binding.     

Attenuated LTP in hippocampal dentate gyrus neurons of mice deficient in the PAF receptor

Platelet-activating factor (PAF), a bioactive lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) derived from phospholipase A(2) and other pathways, has been implicated in neural plasticity and memory formation. Long-term potentiation (LTP) can be induced by the application of PAF and blocked by a PAF receptor (PAF-R) inhibitor in the hippocampal CA1 and dentate gyrus. To further investigate the role of PAF in synaptic plasticity, we compared LTP in dentate granule cells from hippocampal slices of adult mice deficient in the PAF-R and their age-matched wild-type littermates. Whole cell patch-clamp recordings were made in the current-clamp mode. LTP in the perforant path was induced by a high-frequency stimulation (HFS) and defined as >20% increase above baseline of the amplitude of excitatory postsynaptic potentials (EPSPs) from 26 to 30 min after HFS. HFS-induced enhancement of the EPSP amplitude was attenuated in cells from the PAF-R-deficient mice (163 +/- 14%, mean +/- SE; n = 32) when compared with that in wild-type mice (219 +/- 17%, n = 32). The incidence of LTP induction was also lower in the cells from the deficient mice (72%, 23 of 32 cells) than in the wild-type mice (91%, 29 of 32 cells). Using paired-pulse facilitation as a synaptic pathway discrimination, it appeared that there were differences in LTP magnitudes in the lateral perforant path but not in the medial perforant path between the two groups. BN52021 (5 microM), a PAF synaptosomal receptor antagonist, reduced LTP in the lateral path in the wild-type mice. However, neither BN52021, nor BN50730 (5 microM), a microsomal PAF-R antagonist, reduced LTP in the lateral perforant path in the receptor-deficient mice. These data provide evidence that PAF-R-deficient mice are a useful model to study LTP in the dentate gyrus and support the notion that PAF actively participates in hippocampal synaptic plasticity.     

ras and myc analysis in primary and metastatic colorectal carcinomas

Paired primary colorectal adenocarcinoma and normal frozen tissue samples from 60 patients were prospectively studied to determine the frequency of point mutations in K-ras and the occurrence of structural alterations in c-myc. Parallel investigations were performed on liver metastatic specimens from 16 of the patients. 47% of the primary tumors presented point mutations in K-ras; 71% of these were in codon 12. Significant associations were found between codon 13 ras mutations and Dukes' D stage (p<0.05), and between mutations in codon 12 and mucinous type (p<0.01). The c-myc gene structure was altered in 5/60 cases (8%). In 4/16 cases, the K-ras gene status in the primary carcinoma and in the metastatic tissue from the same patient was found to be different. Our results suggest that codon 13 I-as mutations may be associated with an increased invasive and metastatic potential, while codon 12 mutations may have a role in the mucinous differentiation pathway.     

Platelet-activating factor inhibits ionotropic GABA receptor activity in cultured hippocampal neurons

Platelet-activating factor (PAF), one of the most potent bioactive lipids, has been implicated in modulating long-term potentiation (LTP) and neurotoxicity. In the CNS, glutamate and GABA are the major excitatory and inhibitory neurotransmitters, respectively. Previous work has focused on the effects of PAF on glutamatergic receptor responses. The purpose of the present study was to investigate the possible actions of PAF on ionotropic GABA receptor responses in primary cultured hippocampal neurons using the whole-cell and single channel patch clamp techniques. Extracellular application of PAF induced a reduction of the GABA gated Cl- current in a majority of cells (29 of 44 cells), while it caused an enhancement in 10 of 44 cells. A similar heterogeneous modulation of PAF on the GABA receptor activities was also observed in outside-out patch recordings. Moreover, the cell-attached single channel recordings showed that PAF decreased the GABA channel activity. Therefore, PAF may modulate synaptic activity by inhibiting GABA receptor channels. During seizures and neural injury, when enhanced synthesis of this lipid mediator takes place, the actions of PAF on inhibitory GABA receptors may contribute to synaptic dysfunction.     

Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.

PURPOSE: Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury. METHODS: For the in vivo model, epithelium was collected from rabbit corneas at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha regulatory subunit of PI-3K. Two specific PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. RESULTS: Two to four days after in vivo corneal epithelial wound healing, there was a 6-8 fold increase in the expression of the p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85alpha was confined to the wound region and surrounding area. No concomitant increase in PI-3K activity was observed in any of the wound models. Forty-eight hours after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. CONCLUSIONS: Association of most of the increased p85alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.     

Diastematomyelia--a harmless finding or cause of late neurological disturbance? (author's transl)

Diastematomyelia is said to produce neurological lesions. Several new cases of diastematomyelia are reported without any neurological defect. They are discussed and compared with similar cases from the literature. Apparently the fixation of the spinal cord by diastematomyelia seems not to be the primary cause of the "tethering cord syndrome". Other conditions which are sometimes associated with diastematomyelia (myelodysplasia, dysraphia of the spinal cord etc.) may be the primary cause of progressive neurological lesions.     

A threatened privilege

Because their tax-exempt status was at stake, Wisconsin hospitals joined together in 1990 to study and develop a system to better measure and quantify their provision of needed community services. The goal of a task force made up of members of the Catholic Health Association of Wisconsin (CHA-W) and the Wisconsin Hospital Association (WHA) was to develop a proactive response to potential legislative and municipal initiatives that could challenge the tax-exempt status of not-for-profit hospitals. The CHA-W/WHA Task Force on Social Accountability decided to generate data to demonstrate hospitals' tax-exempt worthiness and to show that hospitals pay for many of the direct municipal services they receive. The task force surveyed Wisconsin hospitals on the services they provide to their communities, the municipal service fees they pay, and whether any of their services compete with local businesses. The survey results showed that Wisconsin hospitals do provide needed community services. However, the hospitals do not always adequately communicate to their communities the extent of these benefits. The survey results also showed that Wisconsin hospitals pay most service fees that are quantifiable and measurable. In 1991 the task force adopted a statement of policy which emphasizes that hospitals must clearly demonstrate that they have assessed the health-care needs of their communities, implemented programs to respond to those needs, and maintained their mission to serve.