SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.     

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

The proteolipid protein gene

Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform, DM-20, produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM-20 is also present in myelin-forming and non-myelin-forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM-20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus-Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X-linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM-20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot-Marie-Tooth type in which the peripheral myelin-22 gene is mutated.     

Effects of nitric oxide modulation on tumour blood flow and microvascular permeability in C6 glioma

C6 glioma strongly express nitric oxide synthase. Rats bearing C6 tumours were pre-treated with i.v. Ng-nitro-L-arginine methyl ester (L-NAME), 3-morpholinosydnonimine (SIN-1) or saline before local cerebral blood flow (LCBF) or tumour capillary permeability (TCP) was measured by the [14C]iodoantipyrine autoradiographic or [14C]alpha-amino-isobutyric acid techniques. L-NAME and SIN-1 caused significant TBF alterations (-44% and +136%, respectively) with less marked (-15% and +33%) alterations in normal brain. Calculated cerebrovascular resistance changes within tumour were indeed selective. Baseline TCP was increased compared with normal brain (20-fold). L-NAME and SIN-1 administration did not alter TCP. These effects have significant implications for human malignant glioma management. Selective i.v. manipulation of LCBF, without significant changes in TCP, could increase the efficacy of chemotherapy, radiotherapy or provide better peritumoural oedema control.     

Clinical and biochemical studies in Engelmann's disease (progressive diaphyseal dysplasia)

The clinical and biochemical features of four adults with Engelmann's disease (Camurati-Engelmann disease; progressive diaphyseal dysplasia) are presented. One young patient, with a particularly severe form of the disease, is discussed in detail. Biochemical abnormalities were found in three of the patients. In two of them, one with localized hyperostosis and one with generalized bone disease, the only changes were an increase in the plasma alkaline phosphatase and urinary total hydroxyproline excretion. The most severely affected patients, who had had progressive and generalized bone disease from age two and a half years, also had persistent hypocalcaemia and hyperphosphataemia, a positive calcium balance, and a very low urine calcium excretion. It is suggested that some patients with Engelmann's disease may have a previously unrecognized metabolic disorder associated with increased retention of calcium and excessive bone formation. The possible role of abnormal phosphate metabolism in this increased formation of bone, and the relationship of Engelmann's disease to other hyperostoses, are discussed.     

The effect of norepinephrine on the spinal cord circulation and its possible implications in the pathogenesis of acute spinal trauma

The effect of intra-arterially administered norepinephrine (NE) upon spinal cord blood flow (SCBF), before and after disruption of the blood-cord barrier was studied in dogs. Barrier disruption was accomplished with an intra-arterial bolus injection of 2.5 M urea. Multiple ligations of branches of the posterior aorta and cannula placements ensured that the urea was directed to the lumbar and sacral segments of the cord. The SCBF was measured by the hydrogen clearance method. Intra-arterial urea by itself had no significant effect on SCBF. The intra-arterial infusion of NE (12 microgram/min and 30 microgram/min) was without overall effect on SCBF. However, if the blood-cord barrier had been previously disrupted with hypertonic urea, both concentrations of NE resulted in large reductions in SCBF. No such reductions in SCBF were seen with blood-cord barrier disruption and NE if the animals had been pre-treated with the alpha-blocker, phenoxybenzamine (1.5 mg/kg). Some aspects of the possible involvement of NE in the pathophysiology of acute spinal injury are discussed.     

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.