Basic neural transplantation techniques. I. Dissociated cell suspension grafts of embryonic ventral mesencephalon in the adult rat brain

Campylobacter jejuni is a major pathogen preceding Guillain-Barré syndrome (GBS), and most C. jejuni isolates from GBS patients belong to Penner serotype 19 (heat-stable; HS-19). We analyzed sixteen independent clinical isolates from GBS patients, twelve of which belonged to HS-19, three to HS-2, and one to HS-4, using PCR-based RFLP analysis of a flagellin-A (flaA) gene. Two isolates from patients with Miller Fisher syndrome (MFS), and 27 from patients with uncomplicated enteritis were also examined. All HS-19 isolates, regardless of GBS, showed an identical pattern (Cj-1) by RFLP typing and were distinguishable from those of the other Penner serogroups. In contrast, HS-2 and HS-4 isolates were divided into several different RFLP groups, suggesting HS-19 strains are genetically distinctive among C. jejuni isolates. A DNA fingerprinting method also failed to detect any specific band pattern for GBS-related C. jejuni isolates. We examined relationships among anti-GM1 antibody titres in the sera of GBS patients, clinical forms of GBS, serotype of C. jejuni, and the presence of GM1-like structures in lipopolysaccharide (LPS) components from C. jejuni isolates by immunoblotting. HS-19 related GBS was significantly associated with elevated anti-GM1 antibody titers in the sera of the patients, but not associated with any clinical pattern of GBS. No significant correlations were found between anti-GM1 antibody and the pattern of disease, or between GBS-related C. jejuni strains and the presence of GM1-like structures. HS-19 strains seem to be unique among C. jejuni isolates, and HS-19-related GBS may provide an excellent model for clarification of the pathogenesis of GBS.     

Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation

We have demonstrated the utility of ultrasound backscatter microscopy for targeted intraparenchymal injections into embryonic day (E) 13.5 mouse embryos. This system has been used to test the degree of commitment present in neural progenitors from the embryonic ventral telencephalon and mid-hindbrain region. Many E13.5 ventral telencephalic progenitors were observed to integrate and adopt local phenotypes following heterotopic transplantation into telencephalic or mid-hindbrain targets, whereas mid-hindbrain cells of the same stage were unable to integrate and change fate in the telencephalon. In contrast, many mid-hindbrain cells from an earlier developmental stage (E10.5) were capable of integrating and adopting a forebrain phenotype after grafting into the telencephalon, suggesting that mouse mid-hindbrain progenitors become restricted in their developmental potential between E10.5 and E13.5.     

Peripherally administered tetrahydrobiopterin increases in vivo tryptophan hydroxylase activity in the striatum after transplantation of fetal ventral mesencephalon in six hydroxydopamine lesioned rats

The CD4 receptor of T-helper cells is an essential participant in immune response formation and HIV infection. We report here that the extracellular domains of CD4 receptor can catalyze the phosphotransferase (kinase) reaction. Incubation of rsCD4 in solution with [gamma-32P]ATP results in the Ca2+-dependent autophosphorylation of the protein presumably at a His residue because the reaction is prevented by the diethylpyrocarbonate treatment. The rsCD4 phosphorylates milk casein or human plasma proteins as a Ser/Thr protein kinase.     

Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.     

Differential expression of flectin in the extracellular matrix and left-right asymmetry in mouse embryonic heart during looping stages

The characteristics of six different indicators of response distortion on the Personality Assessment Inventory (PAI; Morey, 1991) were evaluated by having college students complete the PAI under positive impression management, malingering, and honest responding conditions. The six indicators were the PAI Positive Impression (PIM) and Negative Impression (NIM) scales, the Malingering and Defensiveness Indexes, and two discriminant functions, one developed by Cashel and the other by Rogers. Protocols of students asked to malinger were compared with those of actual clinical patients, while protocols of students asked to manage their impression in a positive direction were compared with those of students asked to respond honestly. Comparisons between groups were accomplished through the examination of effect sizes and receiver operating characteristic (ROC) curves. All six indicators demonstrated the ability to distinguish between actual and feigned responding. The Rogers function was particularly effective in identifying malingering. The Cashel function was less effective than other measures in identifying positive impression management, although it appears to also have promise as an indicator of malingering.     

Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.     

Structure and biological activity of the extracellular matrix

The extracellular matrix is formed by complex and intricate networks within which molecules are precisely organized. These molecular networks determine the specific histoarchitecture of tissues and provide cells with information and a scaffold. Most of the structural extracellular matrix molecules - collagens, noncollagenous glycoproteins, and proteoglycans - are chimeric and share common domains. Studies of the interactions between extracellular matrix molecules and mapping of the interaction sites to defined structural modules have led to the concept that the function of the extracellular matrix relies largely in the polymers that they form. Furthermore, determination of the tertiary structure of protein motifs involved either in the assembly of the various molecules into polymers or in cell-extracellular matrix interactions has recently opened the field of structural biology of the extracellular matrix.     

Food intake and lateral hypothalamic self-stimulation covary after medial hypothalamic lesions or ventral midbrain 6-hydroxydopamine injections that cause obesity.

In rats with perifornical lateral hypothalamic (LH) electrodes that induced feeding, self-stimulation through the same electrodes increased immediately after ventromedial hypothalamic (VMH) lesions and did not return to normal until food intake normalized and the rats had become obese. A unilateral far-LH lesion decreased feeding and contralateral perifornical LH self-stimulation. 6-hydroxydopamine (6-OHDA) injected into the midbrain to destroy the ventral noradrenergic bundle (VNAB) caused hyperphagia and increased LH self-stimulation. In summary, VMH or VNAB damage increased feeding and self-stimulation; contralateral far-LH damage decreased both. Results confirm the earlier suggestion that the VMH region is necessary for normal inhibition of feeding and feeding reward as reflected in self-stimulation rate. Although massive 6-OHDA-induced depletion of the dopamine system that passes through the LH can cause starvation and impair self-stimulation, results suggest that selective catecholamine depletion of ventral midbrain neurons with sparing of the A9 and A10 dopaminergic cells can disinhibit feeding and self-stimulation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of focal inactivation of dorsal or ventral layers of the lateral geniculate nucleus on cats' ability to see and fixate small targets

To reveal contributions of different subdivisions of the lateral geniculate nucleus (LGN) to visuomotor behavior, segments of either layer A or the C layers were inactivated with microinjections of gamma-aminobutyric acid while cats made saccades to retinally stabilized spots of light placed either in affected regions of visual space or mirror-symmetric locations in the opposite hemifield. Inactivating layer A reduced the success rate for saccades to targets presented in affected locations from 82.4 to 26.8% while having no effect on saccades to the control hemifield. Saccades to affected sites had reduced accuracy and longer initiation latency and tended to be hypometric. In contrast, inactivating C layers did not affect performance. Data from all conditions fell along the same saccade velocity/amplitude function ("main sequence"), suggesting that LGN inactivations cause localization deficits, but do not interfere with saccade dynamics. Cerebral cortex is the only target of the A layers, so behavioral decrements caused by inactivating layer A must be related to changes in cortical activity. Inactivating layer A substantially reduces the activity of large subsets of corticotectal cells in areas 17 and 18, whereas few corticotectal cells depend on C layers for visually driven activity. The parallels between these behavioral and electrophysiological data along with the central role of the superior colliculus in saccadic eye movements suggests that the corticotectal pathway is involved in both deficits and remaining capacities resulting from blockade of layer A.     

Aging of arterial extracellular matrix elastin: etiology and consequences

In 50 patients with benign ovarian tumours, 39 malignant ovarian carcinoma patients and 39 age-matched healthy women, plasma levels of thrombin-antithrombin III complex and D-dimer were determined as well as CA 125. The coagulation activation marker thrombin-antithrombin III complex and D-dimer levels were elevated in the malignant group compared to the benign and control groups. The results suggest that coagulation and fibrinolysis must play a prominent role in ovarian cancer. Moreover, D-dimer and thrombin-antithrombin III were equally useful as CA 125 for the discrimination of patients with benign or malignant ovarian tumours as evidenced by receiver operating and likelihood ratio calculations.     

Complement activation occurs on subendothelial extracellular matrix in vitro and is initiated by retraction or removal of overlying endothelial cells

Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1beta and TNF-alpha in combination with IFN-gamma markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.     

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower microg l(-1) range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 microg l(-1) urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 microg l(-1) urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.     

Absolute and relative refractory periods of the substrates for lateral hypothalamic and ventral midbrain self-stimulation

The pulse-pair paradigm was used to behaviorally assess the absolute and relative refractory periods of neurons subserving brain-stimulation reward. The amplitude of the second pulse was either equal to, 41% greater than, or 73% greater than the amplitude of the first pulse. In the equal amplitude condition, recovery from refractoriness began as early as 0.4 msec and did not asymptote until as late as 3.5 msec. A 41% increase in the intensity shortened the time course of recovery in five out of six cases. In only one of these five cases did a 73% increase in the intensity of the second pulse produce further changes in time course. Neither increase in the amplitude of the second pulse affected the time course of recovery in one subject. The absolute refractory periods of the directly stimulated reward-relevant neurons appear to be less than 1.5 msec and as short as 0.4 msec; some of these neurons have relative refractory periods that range between 1.0 and 3.5 msec.     

Regulation of survival and death of mesangial cells by extracellular matrix

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.     

Changes in self-stimulation at stimulation-bound eating and drinking sites in the lateral hypothalamus during food or water deprivation, glucoprivation, and intracellular or extracellular dehydration.

Examined the effects of hunger induced by food deprivation, 2-deoxy-{d}-glucose (200 mg/kg), or insulin (2 U/kg) and thirst induced by water deprivation, sodium chloride (4 M), or polyethylene glycol (5 ml of 30% w/w) on lateral hypothalamic self-stimulation in 40 male Long-Evans rats. Changes in self-stimulation were evaluated at electrodes that produced stimulation-bound eating and/or drinking or neither behavior. Daily 30-min test sessions consisted of 3 5-min periods of self-stimulation alternated with 3 5-min periods when barpresses resulted in 5-sec time-out from experimenter-delivered stimulation (stimulation escape). Food deprivation significantly increased self-stimulation; insulin, 2-deoxy-{d}-glucose, and sodium chloride significantly suppressed self-stimulation; water deprivation mildly inhibited self-stimulation; and polyethylene glycol had no effect. This pattern of findings was noted at electrodes that did and those that did not elicit eating and/or drinking. Findings do not support the hypothesis that the magnitude of lateral hypothalamic self-stimulation is differentially and predictably controlled by specific drive mechanisms indexed by the consummatory behaviors also elicited by the stimulation. (41 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Relationship of an extracellular matrix protein, tenascin and breast diseases

Tenascin is an extracellular matrix glycoprotein consisting of six disulfide-linked subunits with molecular masses of 190-250 kDa. Molecular analysis of the tenascin gene revealed that it contains a region homologous to epidermal growth factor genes, repetitive sequences of the type III fibronectin and the fibrinogen gene. Culture studies have shown that tenascin has multiple functions including cell attachment and detachment, promotion and inhibition of neural crest cell migration, cell growth stimulation and hemagglutination. Immunohistochemically, tenascin shows a characteristic and spatially restricted distribution. In mouse mammary glands, tenascin protein is demonstrated in the dense mesenchyme present around growing epithelia during embryogenesis and oncogenesis. Tenascin is expressed in normal human adult breast tissue and benign conditions, although it is expressed more abundantly in breast cancer tissue. Prominent tenascin staining is found in dense cancer-mesenchymal junctions. The staining positivity is significantly correlated with metastasis to regional lymph nodes and tumor grade. Tenascin positive patients have a significantly poorer prognosis compared with tenascin-negative patients. Although the biological functions of tenascin in breast cancer tissue have not yet been clearly elucidated, tenascin staining in surgical tissue specimens might be useful when applied to detect a subgroup of breast cancer patients who have a poorer prognosis.     

Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.     

Extracellular matrix. 2: Role of extracellular matrix molecules and their receptors in the nervous system

Extracellular matrix molecules help regulate many aspects of neural development, including survival, migration, axon growth, and synapse formation by neurons. These same molecules have been shown to modulate regeneration of neurons after injuries. They also regulate the development and differentiation of other neural cells, such as astroglia and Schwann cells. Significant progress has been made recently in characterizing both ECM constituents and their receptors in the nervous system. Extracellular matrix molecules promote cell adhesion, activate intracellular signaling pathways, and modulate the activities of several growth factors and proteins. Our current understanding of the extracellular matrix, its receptors, and its functions in the nervous system are discussed.     

Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.