An Arabidopsis mutant defective in the plastid general protein import apparatus.

Elaborate mechanisms have evolved for the translocation of nucleus-encoded proteins across the plastid envelope membrane. Although putative components of the import apparatus have been identified biochemically, their role in import remains to be proven in vivo. An Arabidopsis mutant lacking a new component of the import machinery [translocon at the outer envelope membrane of chloroplasts (Toc33), a 33-kilodalton protein] has been isolated. The functional similarity of Toc33 to another translocon component (Toc34) implies that multiple different translocon complexes are present in plastids. Processes that are mediated by Toc33 operate during the early stages of plastid and leaf development. The data demonstrate the in vivo role of a translocon component in plastid protein import.

Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis.

The greening phenotypes produced by recessive mutations in a gene designated de-etiolated-2 (DET2) are described. Recessive mutations in the DET2 gene uncouple light signals from a number of light-dependent processes. det2 mutations result in dark-grown Arabidopsis thaliana seedlings with many characteristics of light-grown plants, including hypocotyl growth inhibition, cotyledon expansion, primary leaf initiation, anthocyanin accumulation, and derepression of light-regulated gene expression. In contrast to these morphological and gene expression changes, however, the chloroplast development program is not initiated in the dark in det2 mutants, suggesting that light-regulated gene expression precedes the differentiation of etioplasts to chloroplasts. det2 mutations thus reveal at least two classes of downstream light-regulated responses that differ in their timing and control mechanisms. Homozygous det2 mutations also affect photoperiodic responses in light-grown plants, including timing of flowering, dark adaptation of gene expression, and onset of leaf senescence. The phenotype of det1 det2 double mutants is additive, implying that DET1 and DET2 function in distinct pathways that affect downstream light-regulated genes. Furthermore, these pathways are not utilized solely during early seedling development but must also be required to regulate different aspects of the light developmental program during later stages of vegetative growth.

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants.

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.

Supplementation with estradiol-17beta before the last gonadotropin-releasing hormone injection of the Ovsynch protocol in lactating dairy cows.

The aim of this study was to determine whether an increase in circulating estrogen concentrations would increase percentage pregnant per artificial insemination (PP/AI) in a timed AI protocol in high-producing lactating dairy cows. We analyzed only cows having a synchronized ovulation to the last GnRH of the Ovsynch protocol (867/1,084). The control group (n = 420) received Ovsynch (GnRH--7 d--PGF(2alpha)--56 h--GnRH--16 h--timed AI). The treatment group (n = 447) had the same timed AI protocol with the addition of 1 mg of estradiol-17beta (E2) at 8 h before the second GnRH injection. Ovarian ultrasound and blood samples were taken just before E2 treatment of both groups. In a subset of cows (n = 563), pressure-activated estrus detection devices were used to assess expression of estrus at 48 to 72 h after PGF(2alpha) treatment. Ovulation was confirmed by ultrasound 7 d after timed AI. Treatment with E2 increased expression of estrus but overall PP/AI did not differ between E2 and control cows. There was an interaction between treatment and expression of estrus such that PP/AI was greater in E2-treated cows that showed estrus than in E2-treated or control cows that did not show estrus and tended to be greater than control cows that showed estrus. There was evidence for a treatment by ovulatory follicle size interaction on PP/AI. Supplementation with E2 improved PP/AI in cows ovulating medium (15 to 19 mm) but not smaller or larger follicles. The E2 treatment also tended to improve PP/AI in primiparous cows with low (< or =2.5) body condition score, and in cows at first postpartum service compared with Ovsynch alone. In conclusion, any improvements in PP/AI because of E2 treatment during a timed AI protocol appear to depend on expression of estrus, parity, body condition score, and size of ovulatory follicle.

Prevention of disabling and fatal strokes by successful carotid endarterectomy in patients without recent neurological symptoms: randomised controlled trial.

BACKGROUND: Among patients with substantial carotid artery narrowing but no recent neurological symptom (stroke or transient ischaemia), the balance of surgical risks and long-term benefits from carotid endarterectomy (CEA) was unclear. METHODS: During 1993-2003, 3120 asymptomatic patients with substantial carotid narrowing were randomised equally between immediate CEA (half got CEA by 1 month, 88% by 1 year) and indefinite deferral of any CEA (only 4% per year got CEA) and were followed for up to 5 years (mean 3.4 years). Kaplan-Meier analyses of 5-year risks are by allocated treatment. FINDINGS: The risk of stroke or death within 30 days of CEA was 3.1% (95% CI 2.3-4.1). Comparing all patients allocated immediate CEA versus all allocated deferral, but excluding such perioperative events, the 5-year stroke risks were 3.8% versus 11% (gain 7.2% [95% CI 5.0-9.4], p<0.0001). This gain chiefly involved carotid territory ischaemic strokes (2.7% vs 9.5%; gain 6.8% [4.8-8.8], p<0.0001), of which half were disabling or fatal (1.6% vs 5.3%; gain 3.7% [2.1-5.2], p<0.0001), as were half the perioperative strokes. Combining the perioperative events and the non-perioperative strokes, net 5-year risks were 6.4% versus 11.8% for all strokes (net gain 5.4% [3.0-7.8], p<0.0001), 3.5% versus 6.1% for fatal or disabling strokes (net gain 2.5% [0.8-4.3], p=0.004), and 2.1% versus 4.2% just for fatal strokes (net gain 2.1% [0.6-3.6], p=0.006). Subgroup-specific analyses found no significant heterogeneity in the perioperative hazards or (apart from the importance of cholesterol) in the long-term postoperative benefits. These benefits were separately significant for males and females; for those with about 70%, 80%, and 90% carotid artery narrowing on ultrasound; and for those younger than 65 and 65-74 years of age (though not for older patients, half of whom die within 5 years from unrelated causes). Full compliance with allocation to immediate CEA or deferral would, in expectation, have produced slightly bigger differences in the numbers operated on, and hence in the net 5-year benefits. The 10-year benefits are not yet known. INTERPRETATION: In asymptomatic patients younger than 75 years of age with carotid diameter reduction about 70% or more on ultrasound (many of whom were on aspirin, antihypertensive, and, in recent years, statin therapy), immediate CEA halved the net 5-year stroke risk from about 12% to about 6% (including the 3% perioperative hazard). Half this 5-year benefit involved disabling or fatal strokes. But, outside trials, inappropriate selection of patients or poor surgery could obviate such benefits.

Morphologic and functional correlates of plasma membrane injury during oxidant exposure.

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.

A1 catecholamine cell group: fine structure and synaptic input from the nucleus of the solitary tract.

Preembedding immunoperoxidase staining methods were used to characterize tyrosine hydroxylase-immunoreactive (TH-ir) elements in the caudal ventrolateral medulla, and to determine the extent to which neurons of the A1 cell group are directly innervated by projections of the nucleus of the solitary tract (NTS). TH-ir neurons in the A1 region were medium-sized and multipolar. They possessed rounded nuclei with infrequent invaginations, well-developed Golgi apparati, high cytoplasmic densities of mitochondria, and a low to moderate tendency for rough endoplasmic reticulum (RER) to align in parallel stacks. A1 cell bodies were commonly juxtaposed to TH-positive and TH-negative neurons, myelinated profiles, glia and/or vascular elements, but close membrane appositions were only seen with glial elements. Synaptic input to A1 neurons was predominantly asymmetric, provided virtually exclusively by non-TH-ir terminals, and directed principally to dendritic shafts; A1 somata are relatively sparsely innervated. In a second experiment, silver-intensified immunogold localization of TH-ir was combined with immunoperoxidase labeling for anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), following tracer injections in the caudal aspect of the medial division of the NTS. These experiments revealed a small proportion of PHA-L-labeled axon terminals that made asymmetric contacts with dendritic shafts of TH-ir neurons. These results suggest that the fine structure and synaptic input of A1 neurons are somewhat distinct from that of rostrally situated C1 catecholamine cells. In addition, while they document a direct NTS-A1 projection that may participate in the interoceptive control of vasopressin secretion, the bulk of ventrolaterally directed projections from the caudomedial NTS contact noncatecholaminergic elements in the A1 region, some of which may correspond to so-called depressor neurons implicated in the baroreflex control of sympathetic outflow and vasopressin secretion.

Fine structure and plasticity of barosensitive neurons in the nucleus of solitary tract.

Intravenous phenylephrine (PE) activates neurons in the nucleus of the solitary tract (NTS) whose distribution conforms to those of central projections of the carotid sinus and aortic depressor nerves. This was exploited to permit fine structural characterization of cells presumed to compose the first station in the processing of arterial baroreceptor input, and their responses to stimulation. Rats were perfused at varying intervals after PE injection, and sections through the baroreceptor afferent zone of the NTS prepared for preembedding immunolocalization of Fos-immunoreactivity. Labeled neurons composed a continuous strip extending from the dorsal part of the commissural NTS (NTScom) to the dorsal subnucleus at the level of the area postrema (NTSap). PE-sensitive neurons in these regions were medium-sized, round to ovoid in shape, with scant cytoplasm and an unremarkable complement of organelles. Distinctive features included extensively invaginated nuclei and well-developed Golgi apparati; Fos-ir cells in the NTSap were distinguished from those in NTScom by virtue of better-developed rough endoplasmic reticulum and Golgi, and less convoluted nuclei. Proximal synaptic input to PE-sensitive neurons was sparse and was provided by terminals containing predominantly small, clear synaptic vesicles that formed mainly symmetric junctions with somata and primary dendrites. Prolonged stimulation was accompanied by accentuation of nuclear invaginations, marked accumulation of heterochromatin at their apices, and evidence of enhanced Golgi activity (vesicular budding). These may represent adaptations to facilitate changes in gene expression, to maintain neurotransmitter availability, or both, in the face of a persistent hypertensive challenge.

Ultrastructural localization of the corticotropin-releasing factor-binding protein in rat brain and pituitary.

Preembedding immunoperoxidase staining methods were used to permit ultrastructural analyses of the distribution in rat brain and pituitary of the corticotropin-releasing factor-binding protein (CRF-BP), a moiety distinct from CRF receptors, but which is nonetheless capable of binding the peptide and reversibly neutralizing its biological actions. In anterior pituitary, CRF-BP immunoreactivity (ir) was detected in corticotropelike cells, with reaction product associated principally with secondary lysosomes and multivesicular bodies and not at all with secretory granules. In brain, marked regional differences in the subcellular pattern of CRF-BP staining were evident. In isocortex, where BP/peptide colocalization is rare, BP-ir was distributed in cells and processes in a manner similar to that of a prototypic neuropeptide, including in terminals commonly engaging in synaptic contacts with unlabeled dendritic profiles. In the bed nucleus of the stria terminalis, a site that contains overlapping accumulations of CRF-BP-ir projections and CRF-ir perikarya, BP staining was restricted to vesicle-laden varicosities that rarely engaged in synaptic contacts with somatic or dendritic elements but were frequently apposed to unlabeled axon varicosities and terminals. In the ventromedial medulla, a site of partial CRF/BP overlap, most cells displayed a subcellular localization CRF-BP-ir like that seen in cortex, whereas in others the distribution shared similarities with that observed in pituitary. The results suggest that the function of the CRF-BP may differ in different cellular contexts. In cellular targets of CRF or in neurons in which peptide and BP coexist, the CRF-BP may play a role in processing and degradation of CRF and/or ligand-receptor complexes. In other areas of the central nervous system, the BP seems positioned to serve as a transmitter/modulator at conventional synapses or as an autocrine or paracrine modulator of local CRF effects.

The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization.

In addition to a nonadecapeptide homologous to the teleost melanin-concentrating hormone (MCH), the amino acid sequence predicted from a rat prepro-MCH (ppMCH) cDNA suggested that at least one (neuropeptide EI, or NEI), and possibly a second (NGE), additional neuropeptide may be encoded by this precursor. Cross-reactivity with epitopes of NEI or NGE can account for reported localization of alpha-MSH, rat CRF, and human GRF in rat dorsolateral hypothalamic neurons. We have used antisera raised against rat MCH and NEI in immunohistochemical studies at the light and electron microscopic levels, along with hybridization histochemical localization of ppMCH mRNA, to define the organization of this system. As expected, ppMCH mRNA is prominently expressed in cells in the lateral hypothalamic area and zona incerta. The MCH and NEI peptides were extensively colocalized in neurons in both of these areas. In addition, smaller cell groups in the olfactory tubercle and pontine tegmentum were also positively hybridized for ppMCH mRNA and immunostained for MCH and NEI. Fibers stained for MCH and NEI were similarly, and very broadly, distributed throughout the central nervous system in patterns that generally conformed with known projection fields of the lateral hypothalamic area and zona incerta. A differential distribution was seen in at least one region, the interanterodorsal nucleus of the thalamus, which contained a prominent terminal field stained for MCH but not NEI. At the electron microscopic level, MCH-stained perikarya displayed a prominent staining associated with the Golgi apparatus; this was not encountered in NEI-stained cells. Both peptides were distributed similarly in terminals in the lateral hypothalamic area and median eminence, with staining associated principally with dense-cored vesicles. The results suggest that ppMCH-derived peptides may serve as neurotransmitters or modulators of prominence in a surprisingly expansive projection field of incerto-hypothalamic neurons. The terminal distributions of this system seem most compatible with functional roles in generalized arousal and sensorimotor integration, processes previously implicated as being subject to modulation by the lateral hypothalamic area.

Mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and chloroplast development in Arabidopsis.

When grown in the absence of light, the det1 mutant of Arabidopsis thaliana develops characteristics of a light-grown plant by morphological, cellular, and molecular criteria. Here, we show that recessive mutations at the DET1 locus also result in cell-type inappropriate accumulation of RNAs for light-regulated nuclear and chloroplast genes. det1 root plastids are differentiated into chloroplasts and are present in very high numbers in root cortex cells in contrast to the few starch-containing amyloplasts normally found in Arabidopsis roots. To assay the effect of the det1 mutation on the expression of photoregulated promoters, we used fusion constructs to stably transform wild-type and det1 mutants. We show that the three red-light-regulated chlorophyll a/b binding protein promoters are inappropriately expressed in the roots of det1 seedlings and the blue-light-controlled anthocyanin biosynthetic gene, chalcone synthase, is expressed ectopically in leaf mesophyll cells. These results, together with out previous findings, suggest that the DET1 gene product is a negatively acting regulatory molecule that is used in common by the light stimulus transduction pathway and by temporal or spatial regulatory signals in plants.

The phenotype of Arabidopsis thaliana det1 mutants suggests a role for cytokinins in greening.

When grown in the absence of light, the det1 mutants of Arabidopsis thaliana (L.) Heynh. develop characteristics of light-grown plants as determined by morphological, cellular, and molecular criteria. Further, in light-grown plants, mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and the chloroplast developmental program. Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology. Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally 'light'-regulated. These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate abnormal cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants.

Inhibin/activin subunits are costored with FSH and LH in secretory granules of the rat anterior pituitary gland.

We recently reported that pituitary gonadotropes, major targets of circulating inhibins and activins, are also capable of synthesizing the inhibin (I) alpha- and inhibin/activin (I/A) beta B-subunits. In the present study, we examined the subcellular distribution of these subunits, with special attention given to determinating the extent to which they might be colocalized with the gonadotropins in secretory granules. Pituitaries from adult male rats were cryofixed, molecular distillation-dried, and resin-embedded. Immunogold staining methods were used to examine concurrently the distributions of an I/A subunit and FSH or LH. I/A subunits were detected only in cells that also labeled positively for a gonadotropin, and, in contrast to the gonadotropins, were sequestered almost exclusively within secretory granules. The I alpha-subunit colocalized with FSH in 31%, and with LH in 36%, of all positively stained granules. The I/A beta B-subunit was found with FSH or LH in about 25% of the granules. Approximately 52-69% of the granules contained FSH or LH alone; 7-18% were positive only for an I/A subunit, and this varied as a function of the particular gonadotropin with which costaining was carried out. Dual staining for the I alpha- and the I/A beta B-subunits indicated that at least 35% of all immunolabeled granules showed positive signals for both subunits. Coupled with methodological considerations to indicate that these estimates of the extent of colocalization are likely to be conservative, these data suggest that inhibin and activin are characteristically copackaged, and presumably coreleased, with the gonadotropins.

Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant.

The lipid monogalactosyl diacylglycerol (MGD) is a major structural component of photosynthetic membranes in chloroplasts. Its formation is catalyzed by the enzyme MGD synthase. In many plants, MGD derives from two different biosynthetic pathways: the prokaryotic pathway, which operates entirely within the plastid, and the eukaryotic pathway, which involves steps in the endoplasmic reticulum. Here, we describe the identification and characterization of an Arabidopsis mutant with a defective MGD synthase gene (MGD1). The mutant was identified in a screen of T-DNA lines for individuals with defects in chloroplast biogenesis. It has a yellow-green phenotype that correlates with a approximately 50% deficiency in total chlorophyll per plant. A single T-DNA insertion is located adjacent to the transcription initiation site of the MGD1 gene, and the abundance of MGD1 mRNA is reduced by 75% compared with wild type. Correlation between steady-state MGD1 transcript levels and MGD synthase activity (also reduced by 75% in mgd1) suggests that MGD1 is the most important MGD synthase in green tissues. The amount of MGD in mutant leaves is reduced by 42% compared with wild type. MGD from the mutant contains 23% less 16:3 fatty acid and 10% more 18:3 fatty acid. Because 16:3 is a characteristic feature of MGD from the prokaryotic pathway, it is possible that MGD1 operates with some preference in the prokaryotic pathway. Finally, the MGD-deficiency of mgd1 is correlated with striking defects in chloroplast ultrastructure, strongly suggesting a unique role for MGD in the structural organization of plastidic membranes.

Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light.

The signal transduction pathways that lead to chloroplast biogenesis in plants are largely unknown. We describe here the identification and initial characterization of a novel genetic locus which fits the criteria of a regulatory gene located in a central pathway controlling light-mediated development. In the absence of light, these Arabidopsis thaliana mutants, designated det1 (de-etiolated 1), constitutively display many characteristics that are light-dependent in wild-type plants, including leaf and chloroplast development, anthocyanin accumulation, and accumulation of mRNAs for several light-regulated nuclear and chloroplast genes. The switch between dark and light growth modes thus appears to be a programmed step in a developmental pathway that is defined by det1. We suggest a model where the primary role of light on gene expression is mediated by the activation of leaf development. Further, the recessive nature of the det1 mutation implies that there is negative growth control on leaf development in dicotyledonous plants in the absence of light.

Sulfated carbohydrate compounds prevent microbial adherence by sexually transmitted disease pathogens.

Heparan sulfate (HS) serves as a receptor for adherence of herpes simplex viruses, Chlamydia trachomatis, Neisseria gonorrhoeae, and, indirectly, human immunodeficiency virus. Using primary human culture systems, we identified sulfated carbohydrate compounds that resemble HS and competitively inhibit infection by these pathogens. These compounds are candidates for intravaginal formulations for the prevention of sexually transmitted diseases.

Reliability of radionuclide scintigraphy for detection of testicular torsion: an animal study.

Sodium pertechnetate Tc99m scintigraphy is a valuable technique for the evaluation of acute testicular torsion in postpubescent males. However, in neonates and children with small testicles, the method is less reliable. Since the testicles of adult rats and young children are of similar size, the reliability of testicular imaging for detecting torsion was evaluated in this species. The utility of the radionuclide angiogram (RA) and static images were determined in 17 anesthetized animals before, 2 h after and 20 h after ligation of the left spermatic cord. The preligation RA was asymmetric in 27% of animals, while the static images were abnormal in 18%. Postligation flow and static images were abnormal in 57% and 82% of the animals (localized to the correct side, 38% and 36%), respectively. The animals with vascular occlusion failed to show any statistically significant greater incidence of decreased radionuclide accumulation on the ligated side. To determine the influence of relative perfusion and extracellular fluid space of the scrotum and testicles on the images, additional studies were performed with 201Tl (representing perfusion) and 99mTcO4- (representing ECF space). Perfusion was approximately equal in the testis and epididymis but significantly higher in the scrotum. These results suggest that scrotal scintigraphy is unreliable for detecting acute torsion of small testicles.

The effect of phenothiazines upon maintenance of membrane integrity in the cultured myocardial cell.

The cultured myocardial cell provides a defined model for examining factors which are responsible for maintaining cellular viability and sarcolemmal integrity. Our data indicates that the spontaneous loss of myocyte membrane integrity is a calcium-dependent process and thus provides a method for examining the mechanism through which calcium exerts this effect. Antimyosin antibody staining and propidium iodide uptake were used to quantitate membrane integrity. The integrity of the cell membrane was inversely related to the calcium concentration in the culture medium. This loss of membrane integrity was calmodulin-dependent as demonstrated by the following: phenothiazines (trifluoperazine greater than chlorpromazine greater than promethazine) and structurally dissimilar calmodulin-inhibitors prevented the formation of sarcolemmal defects at concentrations similar to those known to inhibit calmodulin; phenothiazines and calcium demonstrated a competitive interaction with respect to this effect on membrane integrity. Electron microscopy confirmed the integrity of the sarcolemma of the cells exposed to high phenothiazine concentrations although metabolic alterations occurred in these cells as evidenced by an increased membrane permeability to the low molecular weight probe propidium iodide, degenerative changes in the fine structure of the mitochondria, the accumulation of autophagic vacuoles in the cytoplasm and the loss of contractile ability. These findings indicate that calmodulin inhibitory compounds are capable of preserving the membrane integrity of cardiac myocytes, interfering with a calcium-dependent process that is associated with the spontaneous attrition of these cells in culture. Significant intracellular alterations appear at high doses of these agents even while the sarcolemma is free of gross defects.

Sulfated malto-oligosaccharides bind to basic FGF, inhibit endothelial cell proliferation, and disrupt endothelial cell tube formation.

The interaction of basic FGF (bFGF) with heparin, heparan sulfate and related sugars can potentiate or antagonize bFGF activity, depending on the size of the saccharide used. Oligosaccharides based on heparin structures, as small as six sugar residues, have been demonstrated to bind to bFGF and block its activity, while larger structures (> 10 sugar residues) tend to potentiate bFGF. In this study we have synthesized a series of compounds designed to test the requirements of size and sulfation for binding of oligosaccharides to bFGF. These oligosaccharides are not derived from heparin, but rather, are linear chains of glucose linked alpha 1-4 (malto-oligosaccharides) that have been chemically sulfated. In addition to bFGF binding, these compounds were tested for their ability to block basic functions of endothelial cells that are known to be mediated, at least in part, by bFGF. We report that the ability of sulfated malto-oligosaccharides to block binding of bFGF to heparan sulfate was dependent on the size (at least a tetrasaccharide is required), and the degree of sulfation. The activity profile in the bFGF ELISA closely correlated with the ability of these compounds to block REEC or HMVEC tube formation on Matrigel. There was a similar relationship of size and sulfation to the ability of the sulfated malto-oligosaccharides to inhibit endothelial cell growth for most human and rat EC types tested. The single exception was REEC cell growth. One isolate of these cells was stimulated by sulfated malto-oligosaccharides rather than inhibited by them, while a second isolate was neither stimulated nor inhibited. This stimulation showed no correlation with inhibition of bFGF binding in the ELISA assay, suggesting that growth of this cell type was probably not dependent on bFGF. Compounds derived from this series of sulfated, malto-oligosaccharides have the potential to function as bFGF antagonists, are relatively easy to produce, and possess relatively low anticoagulant properties.