Phonons and oxygen diffusion in Bi2O3 and (Bi0.7Y0.3)2O3.

We report investigation of phonons and oxygen diffusion in Bi2O3 and (Bi0.7Y0.3)2O3. The phonon spectra have been measured in Bi2O3 at high temperatures up to 1083 K using inelastic neutron scattering. Ab initio calculations have been used to compute the individual contributions of the constituent atoms in Bi2O3 and (Bi0.7Y0.3)2O3 to the total phonon density of states. Our computed results indicate that as temperature is increased, there is a complete loss of sharp peak structure in the vibrational density of states. Ab initio molecular dynamics simulations show that even at 1000 K in δ-phase Bi2O3, Bi-Bi correlations remain ordered in the crystalline lattice while the correlations between O-O show liquid like disordered behavior. In the case of (Bi0.7Y0.3)2O3, the O-O correlations broadened at around 500 K indicating that oxygen conductivity is possible at such low temperatures in (Bi0.7Y0.3)2O3 although the conductivity is much less than that observed in the undoped high temperature δ-phase of Bi2O3. This result is consistent with the calculated diffusion coefficients of oxygen and observation by quasielastic neutron scattering experiments. Our ab initio molecular dynamics calculations predict that macroscopic diffusion is attainable in (Bi0.7Y0.3)2O3 at much lower temperatures, which is more suited for technological applications. Our studies elucidate the easy directions of diffusion in δ-Bi2O3 and (Bi0.7Y0.3)2O3.

White matter protection with insulin-like growth factor 1 and hypothermia is not additive after severe reversible cerebral ischemia in term fetal sheep.

Moderate cerebral hypothermia significantly improves survival without disability from perinatal hypoxia-ischemia. However, protection is partial. Insulin-like growth factor 1 (IGF-1) plays a key role in oligodendrocyte survival and myelination. The purpose of this study was to test the hypothesis that the combination of IGF-1 plus hypothermia could reduce postischemic white matter damage compared with hypothermia alone. Unanesthetized near-term fetal sheep received 30 min of cerebral ischemia, followed by either an infusion of 3 µg of IGF-1 intracerebroventricularly from 4.5 to 5.5 h plus cooling from 5.5 to 72 h (IGF-1 + hypothermia; n = 8), vehicle infusion plus cooling from 5.5 to 72 h (vehicle + hypothermia; n = 12), sham cooling plus sham infusion (ischemia control; n = 12) or sham ischemia (n = 5). The fetal extradural temperature was reduced from 39.4 ± 0.1°C to between 30 and 33°C. White matter was assessed after 5 days. Ischemia was associated with severe loss of CNPase-positive oligodendrocytes in white matter compared with sham ischemia (380 ± 138 vs. 1,180 ± 152 cells/field; mean ± SD; p < 0.001). Delayed hypothermia reduced cell loss (847 ± 297 cells/field, p < 0.01, vs. ischemia control), but there was no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (1,015 ± 211 cells/field; NS). Ischemia was associated with increased caspase 3 expression in white matter (216 ± 41 vs. 19 ± 18 cells/field; p < 0.001). Hypothermia reduced numbers of activated caspase 3-positive cells (116 ± 81 cells/field; p < 0.05), with no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (91 ± 27 cells/field; NS). In conclusion, delayed cotreatment with IGF-1 plus hypothermia after ischemia was associated with an improvement in white matter damage similar to that achieved by hypothermia alone.

Encapsulated living choroid plexus cells: potential long-term treatments for central nervous system disease and trauma.

In neurodegenerative disease and in acute brain injury, there is often local up-regulation of neurotrophin production close to the site of the lesion. Treatment by direct injection of neurotrophins and growth factors close to these lesion sites has repeatedly been demonstrated to improve recovery. It has therefore been proposed that transplanting viable neurotrophin-producing cells close to the trauma lesion, or site of degenerative disease, might provide a novel means for continuous delivery of these molecules directly to the site of injury or to a degenerative region. The aim of this paper is to summarize recent published information and present new experimental data that indicate that long-lasting therapeutic implants of choroid plexus (CP) neuroepithelium may be used to treat brain disease. CP produces and secretes numerous biologically active neurotrophic factors (NT). New gene microarray and proteomics data presented here indicate that many other anti-oxidant, anti-toxin and neuronal support proteins are also produced and secreted by CP cells. In the healthy brain, these circulate in the cerebrospinal fluid through the brain and spinal cord, maintaining neuronal networks and associated cells. Recent publications describe how transplanted CP cells and tissue, either free or in an immunoprotected encapsulated form, can effectively deliver therapeutic molecules when placed near the lesion or site of degenerative disease in animal models. Using simple techniques, CP neuroepithelial cell clusters in suspension culture were very durable, remaining viable for 6 months or more in vitro. The cell culture conditions had little effect on the wide range and activity of genes expressed and proteins secreted. Recently, completed experiments show that implanting CP within alginate-poly-ornithine capsules effectively protected these xenogeneic cells from the host immune system and allowed their survival for 6 months or more in the brains of rats, causing no adverse effects. Previously reported evidence demonstrated that CP cells support the survival and differentiation of neuronal cells in vitro and effectively treat acute brain injury and disease in rodents and non-human primates in vivo. The accumulated preclinical data together with the long-term survival of implanted encapsulated cells in vivo provide a sound base for the investigation of these treatments for chronic inherited and established neurodegenerative conditions.

Formulating the alginate-polyornithine biocapsule for prolonged stability: evaluation of composition and manufacturing technique.

Alginate encapsulation is one of the most widely used techniques for introducing cell-based therapeutics into the body. Numerous encapsulation methodologies exist, utilizing a variety of alginates, purification technologies, and unique polycationic membrane components. The stability of a conventional alginate formulation encapsulated using a commercially available technique and apparatus has been characterized extensively. The current study employs an encapsulation protocol and ultra-pure alginate pioneered at the University of Perugia. The enhanced microcapsules were produced, characterized, and implanted into the brain, peritoneal cavity, and subcutaneous space of Long-Evans rats. After 14, 28, 60, 90, 120, and 180 or 215 days, capsules were explanted and the surface was analyzed using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Image analysis was carried out to measure changes in diameter and wall thickness. FTIR peak analysis and surface morphology from SEM indicated that the enhanced encapsulation technique and formulation produced a stable biocapsule capable of survival in all sites, including the harsh peritoneal environment, for at least 215 days. Preimplant analysis showed a marked increase in the structural integrity of the enhanced formulation with improved elasticity and burst strength compared with the baseline formulation, which remained stable for less than 60 days. The enhanced microcapsule composition showed advantages in physical strength and longevity, indicating that small changes in encapsulation methodologies and materials selection can dramatically impact the stability and longevity of alginate microcapsules and their contents.

Morphology and development of the reticuloperidial ascomata of Auxarthron conjugatum.

Light and electron microscopy showed that the reticuloperidium of thick-walled hyphae, characteristic of the mature ascoma of Auxarthron conjugaturn, originated from branches that grew from the broad, gyre-like hyphal loops making up the ascomatal initials. Within the developing peridium, short, acropetally proliferating chains of prototunicate asci each arose from a single crozier and matured from base to tip. The walls of young asci were two-layered but evanesced as they matured with the outer layer dissolving before the inner one. Distal asci in some chains retained the inner wall, detached from adjacent asci by septum schizolysis and when transferred to fresh media produced germ tubes and mycelium. Ultraviolet epifluorescent staining with a DNA intercalator (Hoechst) indicated that these spore-like asci probably contained diploid nuclei. In normal asci, ascospores had an inner, electron lucent primary wall and a three-layered secondary wall. The deposition pattern of the middle layer of the secondary wall created the distinctive array of pits and ridges characteristic of the ascospores in this taxon. The production of ascospores, spore-like asci and arthroconidia, along with the tendency of ascospores to adhere in a mass, is interpreted as contributing to the reproductive flexibility and inoculum potential of A. conjugatum. In all respects the ascomata of A. conjugatum differed substantially from the morphologically similar taxon, Myxotrichum arcticum. These findings underscore the benefit of using DNA-based phylogenies in concert with cytological and ultrastructural observations for exploring selective pressures behind homoplasious characters and revealing novel structural features.

CNS grafts of rat choroid plexus protect against cerebral ischemia in adult rats.

The present study examined the neuroprotective effects of choroid plexus isolated from adult rats and encapsulated within alginate microcapsules. In vitro, conditioned media from cultured choroid plexus produced a marked, dose-dependent protection of embryonic cortical neurons against serum deprivation-induced cell death. In vivo studies demonstrated that a one-hour middle cerebral artery occlusion in adult Wistar rats produced profound motor and neurological impairments 1-3 days after stroke. In contrast, stroke animals transplanted with encapsulated choroid plexus cells displayed a significant reduction in both motor and neurological abnormalities. Histological analysis 3 days post-transplantation revealed that choroid plexus transplants significantly decreased the volume of striatal infarction. This is the first report demonstrating the therapeutic potential of transplanted choroid plexus for stroke.

Immunohistochemical staining for Chlamydia pneumoniae is increased in lung tissue from subjects with chronic obstructive pulmonary disease.

The seroprevalence of Chlamydia pneumoniae is increased in chronic obstructive pulmonary disease (COPD), and subjects with COPD are more likely to have a positive polymerase chain reaction for C. pneumoniae in their sputum. It has been suggested that C. pneumoniae may have a role in the pathogenesis of COPD. We undertook immunohistochemistical staining for C. pneumoniae in archival tissue from subjects who had undergone lobectomy for bronchial carcinoma. There were 16 subjects with COPD (FEV(1) = 64 +/- 8% [mean +/- SD] predicted) and 21 subjects with normal lung function (FEV(1) = 95 +/- 11% predicted). There was no difference between the two groups in age or smoking history. Tissues from all of the subjects stained positively for C. pneumoniae, but in the subjects with COPD there were 14.5 positive cells per field (magnification x400), as compared with 9.3 cells per field in the control subjects (p = 0.02). Fifty-four percent of the macrophages from the subjects with COPD stained positively for C. pneumoniae, as compared with 29% from the control subjects (p < 0.001). A second control group consisted of 18 younger individuals (mean age: 32 yr) who died accidentally. Only 44% of these subjects had positive staining for C. pneumoniae, and the mean number of cells per field was 0.4. These findings suggest that persistent infection with C. pneumoniae is common, and that there is increased immunostaining for C. pneumoniae in COPD. Further studies are necessary to determine whether chronic infection with C. pneumoniae is important in the pathogenesis of COPD.

Intracerebral transportation and cellular localisation of insulin-like growth factor-1 following central administration to rats with hypoxic-ischemic brain injury.

Insulin-like growth factor-1 (IGF-1) has been shown to be neuroprotective when administered centrally following hypoxic-ischemic (HI) brain injury. However, the cerebral distribution and site of action of IGF-1 after intracerebroventricular (i.c.v.) administration are not known. A unilateral HI brain injury was induced in adult rats by a modified Levine method. Either 3H-IGF-1 alone, or in combination with unlabelled IGF-1, was administered into the lateral ventricle 2 h after injury. The activity of 3H-IGF-1 signal in the potentially injured cortex was compared between two treatment groups using image analysis. The regional distribution and cellular localisation of 3H-IGF-1 were examined autoradiographically in potentially injured hemispheres at 0.5 and 6 h after administration. Tritiated IGF-1 was detected predominantly in the pia mater, perivascular spaces and subcortical white matter tracts 0.5 h after administration and decreased by 6 h (p<0.05). The signals associated with the perivascular spaces and pia mater were not blocked by unlabelled IGF-1, suggesting non-saturable binding in these brain areas. IGF-1 signal was co-localised with IGF binding protein (IGFBP)-2 immunostaining in the white matter tracts where the signal was blocked by unlabelled IGF-1, suggesting competitive association. IGF-1 signal associated with neurons and glia was maximal in the cerebral cortex and less in the CA1-2 subregion of the hippocampus which were blocked by unlabelled IGF-1 (p<0.05). The signals from cortical neurons did not decrease 6 h after administration, suggesting specific and persistent binding to these cells. Our results indicate that centrally administered IGF-1 can be translocated to neurons and glia via the perivascular circulation and the ependymal cell-white matter tract pathways.

Impaired insulin-mediated glucose uptake in monocytes of short children with intrauterine growth retardation.

AIMS: To determine whether there was an impairment in insulin-mediated glucose uptake in monocytes from short children with intrauterine growth retardation (IUGR) when compared with control subjects. METHODS: Circulating monocytes were isolated by histopaque gradient separation followed by adherence. Monocytes were incubated with insulin at the following concentrations; 0, 0.1, 0.2, 0.6, 1, 2 and 6 nm. 2-deoxyglucose (2-DG) uptake was measured after incubation with [(3)H]2-DG and expressed as pmol/min/10(6) cells. Insulin-stimulated glucose uptake was determined in two ways: 6 nm insulin concentration minus baseline (6-0 nm) and the regression slope of glucose uptake over the range of log insulin concentrations (slope value). Insulin sensitivity was determined from a 90-min frequently sampled intravenous glucose tolerance test with the minimal model. RESULTS: Short children with IUGR (n = 16) had lower slope (4.6 +/- 1.1 vs. 9.5 +/- 2.0, p = 0.002) and 6-0 nm (8 +/- 2 vs. 15 +/- 3 pmol/min/10(6) cells, p = 0.048) glucose uptake values than normal children (n = 11). There was no difference in baseline glucose uptake between IUGR and normal children (36 +/- 5 vs. 48 +/- 7 pmol/min/10(6) cells). In the five subjects with IUGR that were evaluated, the in vivo insulin sensitivity index and glucose effectiveness were found to be positively correlated with insulin-mediated glucose uptake in monocytes (r = 0.54) and baseline glucose uptake in monocytes, respectively (r = 0.69). CONCLUSIONS: Short children with IUGR have impairment in insulin-mediated glucose uptake in monocytes when compared with normal children. Our hitherto limited data indicate that insulin-mediated glucose uptake in monocytes is correlated with in vivo assessment of insulin sensitivity in children with IUGR.

Key enzymes of prostaglandin biosynthesis and metabolism. Coordinate regulation of expression by cytokines in gestational tissues: a review.

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.

Neuroprotective effects of Gly-Pro-Glu, the N-terminal tripeptide of IGF-1, in the hippocampus in vitro.

Insulin-like growth factor 1 (IGF-1) plays a critical role in CNS development. IGF-1 can block neuronal apoptosis in vitro and in vivo. IGF-1 is thought to be cleaved into des-N-(1-3)-IGF-1 and an amino terminal glycine-proline-glutamate (GPE tripeptide). Here we report a neuroprotective role for GPE tripeptide, with enhanced survival of the CA1-2 hippocampal neurons following an excitotoxic insult in vitro. Binding and displacement studies suggest uniquely distributed sites of action within the rat including the hippocampal CA1-2, pyriform cortex, amygdala, choroid plexus, blood vessels and to a lesser extent in the cortical regions. A similar pattern of binding was seen in the human. This finding could lead to new strategies to reduce neuronal death after injury and in disease.

Effects of maternal captopril treatment on growth, blood glucose and plasma insulin in the fetal spontaneously hypertensive rat.

In the spontaneously hypertensive rat (SHR) fetal growth and metabolism are abnormal. It has been speculated that maternal hypertension may be the cause of these abnormalities. Captopril treatment, which reduces maternal blood pressure, during pregnancy and lactation, is reported to have a beneficial effect postnatally, normalizing the blood pressure of offspring in the SHR. In the present study, the effects of maternal captopril treatment on fetal growth and plasma metabolites were investigated in the fetuses of two rat strains (SHR and Wistar-Kyoto (WKY)), in order to determine whether normalizing maternal blood pressure also normalized abnormalities in fetal growth and metabolism. On fetal Day 20, SHR fetuses were lighter and placentae were heavier than for the corresponding WKY. Captopril had no effect on fetal weight in the SHR, but decreased it in the WKY. There was no effect of captopril on placental weight. Fetal plasma insulin levels were higher in the SHR than in the WKY and were decreased by captopril treatment in both strains. Fetal blood glucose was elevated and fetal blood lactate was decreased in captopril-treated litters from both strains. Captopril had no effect on fetal plasma IGF-1 but fetal plasma IGF-2 levels were lower in the captopril-treated SHR than in the captopril-treated WKY. These findings suggest that maternal captopril treatment decreases insulin secretion in the fetal rat. High levels of fetal plasma insulin suggest that the SHR fetus is insulin resistant. Fetal insulin levels may contribute to the adverse consequences of gestational captopril treatment observed in many species. The differences in the effect of captopril on the two strains suggest that there are underlying endocrine differences in the SHR.

Fetal rat lung epithelium has a functional growth hormone receptor coupled to tyrosine kinase activity and insulin-like growth factor binding protein-2 production.

Although growth hormone (GH) receptor (GHR) mRNA and protein are present in fetal tissues such as the lung, there is little evidence that GH mediates growth in the fetus. We have identified functional responses to GH in fetal rat lung epithelia and suggest a possible role for GHR in the developing lung. GHR mRNA in lung extracts was high before birth at day 16 of gestation (16f), decreased to low levels at day 22f but increased again after birth. At day 20f GHR mRNA levels were higher in lung than in liver, whereas growth hormone binding protein mRNA levels were approximately equal in lung and liver. Stimulation of primary cell cultures of day 19f lung epithelia with GH caused increased tyrosine phosphorylation in specific proteins, demonstrating functional GHR. Lung fibroblasts isolated at the same time did not respond to GH. Ligand and Northern blot analysis of the epithelial cultures revealed that GH stimulation increased insulin-like growth factor binding protein-2 (IGFBP-2) activity and mRNA. These experiments demonstrate the functional activity of GHR, specifically in fetal lung epithelium. We suggest that one role for GH in vivo may be indirectly to modify insulin-like growth factor activity in the developing fetal lung by increasing IGFBP-2.

Fetal and placental glucose and amino acid uptake in the spontaneously hypertensive rat.

Until late in gestation, fetal spontaneously hypertensive rat (SHR) is growth retarded. Fetal growth rate increases after placental hypertrophy occurs between fetal days 18 and 20. The increase in placental mass may result in improved transfer of macro nutrients to the fetus and thus stimulate growth. In this study, fetal and placental uptake of the glucose analog 3-O-methyl glucose (3MG) and the amino acid analog amino-isobutyric acid (AIB) from the maternal circulation were compared in the SHR and Wistar-Kyoto (WKY) on days 16, 20 and 22 of gestation. Placental 3MG uptake (d/min/g tissue wet weight) was decreased in the SHR on days 20 and 22 but no differences were observed in fetal 3MG uptake. Increased placental mass in the SHR meant that total placental 3MG uptake was greater in the SHR. Placental uptake of AIB (d/min/g tissue wet weight) was much lower in the SHR (on days 16, 20 and 22) and the decrease was not compensated for by increased placental mass. Fetal uptake of AIB was decreased on days 20 and 22 (P<0.05). AIB uptake (d/min/g tissue wet weight) by the carcass and the internal organs (brain, heart, kidney, liver and lung) was also lower in the SHR. These findings indicate that although fetal growth in the SHR increases rapidly in late gestation following placental hypertrophy, it does so despite a pronounced deficit in amino acid uptake.

Glucocorticoid activity in the fetal spontaneously hypertensive rat.

Fetal exposure to high concentrations of corticosteroids in the rat is associated with elevated blood pressure in postnatal life. In this study we have investigated indicators of corticosteroid activity in fetal spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in order to determine whether fetal corticosteroid exposure is increased in the SHR. Placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity, which prevents maternal steroids from crossing the placenta, was not impaired in the SHR. Concentrations of amniotic fluid corticosterone were significantly decreased in the SHR compared with the WKY at fetal Day 20, but were not significantly different on fetal Days 16 or 22. This suggests that rather than increased exposure to corticosteroids in the SHR fetus corticosteroid exposure may be reduced. Expression of lung surfactant protein A (Sp-A), a gene induced in late gestation by corticosteroids, was decreased in the SHR. In addition, differences in amniotic fluid electrolyte concentrations were observed which may reflect delayed renal maturation in the fetal SHR. These data suggest that the SHR fetus is exposed to low concentrations of corticosteroids and that the late gestation rise in fetal corticosteroid may be delayed in the SHR.

Perinatal growth disturbance in the spontaneously hypertensive rat.

Disproportionate fetal and placental growth are associated with the development of hypertension in the rat and human. Here we report differences in fetal, neonatal, and placental growth, and in metabolism and endocrinology, between the spontaneously hypertensive rat (SHR), a genetic model for human essential hypertension, and the control Wistar-Kyoto (WKY) strain. Gestation in SHR (23 d) was longer than in WKY by 20 h. Body weights were lower in the SHR from fetal d 16 to 20 and on postnatal d 15. However, on fetal d 22 and postnatal d 1, there was no significant difference in body weight between SHR and WKY. SHR placentas were larger than those of WKY at d 20, and by term there was a difference of 30% (p < 0.01). Other indices of disproportionate growth were hypertrophy of the fetal heart and kidney and decreased ponderal index in the SHR neonate. Blood glucose in SHR fetuses was lower than in WKY fetuses (p < 0.05), whereas blood lactate was higher (p < 0.05) and fetal hematocrit was reduced (p < 0.001). These findings suggest undernutrition and placental insufficiency may occur in SHR fetuses. Plasma IGF-II was increased on the last day of gestation in both strains, whereas IGF-I was unaltered. Fetal liver IGFBP-2 mRNA and plasma IGFBP-2 levels were reduced in SHR on fetal d 20 and 22 (p < 0.01). Differences in growth and endocrine and metabolic parameters suggest abnormal perinatal physiology in the SHR, which may influence the later development of hypertension.

Response of airway smooth muscle cells to TGF-beta 1: effects on growth and synthesis of glycosaminoglycans.

Transforming growth factor-beta (TGF-beta) is formed in the airways and may have a role in airway remodeling in asthma. We have studied the effects of TGF-beta on bovine airway smooth muscle cells (BASMC) in vitro. Thymidine incorporation by BASMC was inhibited after a 24-h incubation with TGF-beta 1. In contrast, thymidine incorporation by BASMC was stimulated (35.1 +/- 11.2%) after a 48-h incubation with 1 ng/ml TGF-beta 1. Cell number was also increased (25.9 +/- 7.6%) after a 72-h incubation with 3 ng/ml TGF-beta 1. TGF-beta 1 also increased cell size at 72 h, with a 24.3 +/- 6.2% increase in cell, diameter. Increases in BASMC size were accompanied by increased [3H]proline incorporation into cell protein. In cells from any individual animal, there was a strong inverse correlation (r = -0.97) between changes in cell number and cell size. In cells from some animals, the main effect of TGF-beta 1 was to promote an increase in cell number, whereas in others the predominant effect was cell hypertrophy. In contrast epidermal growth factor (EGF) led to an increase in thymidine incorporation and cell number in all preparations but did not increase cell size. TGF-beta 1 also promoted secretion of glycosaminoglycans into culture medium by BASMC with a preferential increase in hyaluronan secretion (4.5-fold) after 24 h. Latent TGF-beta (0.89 +/- 0.06 ng/ml) was also detected in conditioned medium from cultured BASMC, and TGF-beta 1 expression was demonstrated with RNA extracts from BASMC. Varying degrees of both smooth muscle cell hypertrophy and hyperplasia occur in asthma. These results obtained with airway smooth muscle cells indicate that TGF-beta could play a role in the structural changes seen in asthma.

Cloning of novel kinectin splice variants with alternative C-termini: structure, distribution and evolution of mouse kinectin.

The analysis of cDNA clones encoding novel variant forms of mouse kinectin, an endoplasmic reticulum (ER)-bound receptor for the motor protein kinesin, is reported. Kinesin and cytoplasmic dynein are involved in mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduced from kinectin cDNA isolated from mouse spleen cell and testis libraries revealed a long signal peptide or transmembrane sequence, and a 328 amino acid residue globular N-terminal domain adjacent to a much larger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple contiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) insertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. Insert 5 contains a termination codon, producing a C-terminus that is highly homologous to that of human kinectin. Three out of five mouse kinectin clones lack insert 5, generating a novel eleven amino acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional relevance given that the C-termini are exposed for interaction with kinesin, whereas the globular N-terminus is embedded in the ER membrane. Alternative C-termini represent candidate modifications that could determine specificity of binding to kinesin or cytoplasmic dynein, and the switching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, which provides the first indication that the kinectins are very widely distributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine homologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse kinectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues. There is a two-fold molar excess of kinectin over kinesin in unextracted vesicles, suggesting that kinectin might be a dimer. The electrostatic properties of the coiled-coil region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support this notion.

The effects of insulin-like growth factor (IGF)-1, IGF-2, and des-IGF-1 on neuronal loss after hypoxic-ischemic brain injury in adult rats: evidence for a role for IGF binding proteins.

Insulin-like growth factor (IGF)-1, IGF binding protein (IGFBP)-2, and IGFBP-3 are expressed in the rat brain in regions of neuronal loss by 3 days after hypoxic- ischemic (HI) brain injury and IGF-2 somewhat later. Central administration of rh-IGF-1 after HI injury reduces neuronal loss in vivo. To clarify the mode of action of IGF-1 and the potential role of IGFBPs, the effects of IGF-1, IGF-2, des(1-3)-N-IGF-1 (des-IGF-1), an analogue of IGF-1 with low affinity for IGFBPs, and IGF-1 combined with IGF-2 were compared 2 h after administration into the lateral cerebral ventricle after an HI injury. Unilateral HI was induced in adult rats by right carotid artery ligation followed by 10- min exposure to 6%O2. The extent of neuronal loss was determined in the cortex, striatum, hippocampus, dentate gyrus, and thalamus 5 days later. Central administration of 20 micrograms IGF-1 (n = 17) reduced neuronal loss in all regions (P < 0.01). Neither 20 micrograms IGF-2 (n = 17), 2 micrograms des-IGF-1 (n = 10), nor 20 micrograms des-IGF-1 (n = 17) reduced neuronal loss. There was a trend towards a reduction in neuronal loss after 150 micrograms des-IGF-1 (n = 20). IGF-2 alone increased neuronal loss in the hippocampus and dentate gyrus compared with the same regions in vehicle-treated animals (P < 0.05). Coadministration of 30 micrograms IGF-2 blocked the neuroprotective effects of 20 micrograms IGF-1 (n = 18, P < 0.05) and reduced the accumulation of [3H]IGF-1 in the injured hemisphere (n = 4) (P < 0.05). These observations suggest a role for IGFBPs in targeting the neuroprotective actions of IGF-1. IGF-2 may antagonize the protective effect of IGF-1 by displacing it from IGFBPs.