Measurement of the Spin Absorption Anisotropy in Lateral Spin Valves.

The spin absorption process in a ferromagnetic material depends on the spin orientation relative to the magnetization. Using a ferromagnet to absorb the pure spin current created within a lateral spin valve, we evidence and quantify a sizable orientation dependence of the spin absorption in Co, CoFe, and NiFe. These experiments allow us to determine the spin-mixing conductance, an elusive but fundamental parameter of the spin-dependent transport. We show that the obtained values cannot be understood within a model considering only the Larmor, transverse decoherence, and spin diffusion lengths, and rather suggest that the spin-mixing conductance is actually limited by the Sharvin conductance.

Giant magnetoresistance in lateral metallic nanostructures for spintronic applications.

In this letter, we discuss the shift observed in spintronics from the current-perpendicular-to-plane geometry towards lateral geometries, illustrating the new opportunities offered by this configuration. Using CoFe-based all-metallic LSVs, we show that giant magnetoresistance variations of more than 10% can be obtained, competitive with the current-perpendicular-to-plane giant magnetoresistance. We then focus on the interest of being able to tailor freely the geometries. On the one hand, by tailoring the non-magnetic parts, we show that it is possible to enhance the spin signal of giant magnetoresistance structures. On the other hand, we show that tailoring the geometry of lateral structures allows creating a multilevel memory with high spin signals, by controlling the coercivity and shape anisotropy of the magnetic parts. Furthermore, we study a new device in which the magnetization direction of a nanodisk can be detected. We thus show that the ability to control the magnetic properties can be used to take advantage of all the spin degrees of freedom, which are usually occulted in current-perpendicular-to-plane devices. This flexibility of lateral structures relatively to current-perpendicular-to-plane structures is thus found to offer a new playground for the development of spintronic applications.

Comparison of the use of NiFe and CoFe as electrodes for metallic lateral spin valves.

Spin injection and detection in Co60Fe40-based all-metallic lateral spin valves have been studied at both room and low temperatures. The obtained spin signals amplitudes have been compared to those of identical Ni80Fe20-based devices. The replacement of Ni80Fe20 by CoFe allows increasing the spin signal amplitude by up to one order of magnitude, thus reaching 50 mΩ at room temperature. The spin signal dependence with the distance between the ferromagnetic electrodes has been analyzed using both a 1D spin-transport model and finite element method simulations. The enhancement of the spin signal amplitude when using CoFe electrodes can be explained by a higher effective polarization.

Diagnostic heterogeneity of diabetes in lean young adults: classification based on immunological and genetic parameters.

The aim of our study was to investigate the relative prevalence of the different forms of diabetes in young adults and their respective clinical characteristics. Included were 51 nonobese patients (BMI < 27 kg/m2) with diabetes diagnosed before age 40, excluding typical IDDM. Each patient was subjected to screening for glucokinase gene (MODY2) and mitochondrial DNA (at nucleotide 3243) mutations, to HLA class II genotyping, and screening for the presence of islet cell antibodies (ICAs) and anti-GAD antibodies. Informative families were analyzed for linkage of diabetes to chromosome 12q (MODY3). Based on clinical criteria, patients were subdivided into MODY (n = 19) and non-MODY (n = 32). In the MODY group, we identified three patients with MODY2, one with the 3243 mitochondrial mutation, and another with autoimmune diabetes. One of the five MODY families available for linkage study was shown to have MODY3. In the non-MODY group, we found five patients with autoimmune diabetes and one with MODY2. No clinical parameter was helpful to classify patients in one of these subclasses of diabetes; however, the glucagon-stimulated C-peptide was useful to discriminate between MODY2 patients and the others. In conclusion, young and lean non-insulin-dependent diabetic patients constitute a very heterogeneous group, although they present similar clinical characteristics. The clinical distinction of MODY and non-MODY patients allows correct classification in, at most, 75% of the patients and thus is not sufficient to predict clinical course. However, immunological and genetic parameters allowed us to classify only 25% of the patients in specific diagnostic classes.

Abnormal regulation of hepatic glucose output in maturity-onset diabetes of the young caused by a specific mutation of the glucokinase gene.

A subtype of maturity-onset diabetes of the young (MODY) is caused by mutations of the glucokinase gene, an enzyme expressed in pancreatic beta-cells and the liver. To assess the consequences of a functional alteration of glucokinase at the level of the liver, endogenous (hepatic) glucose production and glucose cycling (an indirect assessment of hepatic glucokinase activity) were measured with 2-2H glucose and 6,6-2H glucose in patients who developed MODY because of the V203A mutation of glucokinase, and in control subjects at similar levels of glycemia. Measurements were performed in the postabsorptive state and after ingestion of 13C-labeled glucose. In the postabsorptive state, MODY patients had normal glucose production (10.9 +/- 1.3 vs. 11.3 +/- 0.6 micromol x kg(-1) x min(-1)) but decreased glucose cycling (0.6 +/- 0.3 vs. 1.5 +/- 0.3 micromol x kg(-1) x min(-1); P < 0.05) when compared with control subjects. However, at plasma glucose and insulin levels similar to those observed in MODY patients, control subjects' glucose production was markedly lower (3.2 +/- 1.5 micromol x kg(-1) x min(-1). After glucose ingestion, endogenous glucose production was reduced by only 29% in MODY patients compared with 80% in control subjects at a similar level of hyperglycemia (P < 0.05). This suggests that the V203A mutation of glucokinase results in decreased activity of glucokinase in liver cells. Thus endogenous glucose production is inadequately inhibited by hyperglycemia in MODY patients, possibly as a result of impaired hepatic glucokinase activity. These alterations contribute to the pathogenesis of hyperglycemia.

Hypoglycaemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy.

We describe a 48-year-old woman with recurrent severe hypoglycaemia apparently caused by a paraprotein with insulin-binding capacity. Very high fasting values were found for serum insulin (170 and > 250 mU l-1) as well as for proinsulin 125 pmol l-1 and an insulinoma was suspected. Hypoglycaemia developed after an oral glucose tolerance (OGTT) test but not during fasting for 48 h. Free insulin and C-peptide were normal during OGTT whereas serum insulin was very high. 125I-insulin binding to serum, determined with a polyethylene glycol (PEG) precipitation method was high (40%), and equally high after addition of 1.7 x 10(-5) mol l-1 cold insulin to estimate non-specific binding. By adding very high concentrations of cold insulin, displacement of 125I-insulin bound to serum was found (50% displacement at 4 x 10(-5) mol l-1). No immunoglobulin G (IgG) insulin antibodies were detected by radioimmunoelectrophoresis. On agarose electrophoresis a small paraprotein (4 g l-1) in the gamma-globulin fraction was detected. 125I-insulin binding to this paraprotein was demonstrated. We conclude that if insulin autoantibodies are suspected as a cause of hypoglycaemia screening for insulin antibodies should always be done with a PEG-precipitation method.

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies.

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.

Monitoring receptor mediated regulation of cytosolic calcium in single pituitary cells by dual excitation microfluorimetry.

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.

Single cell monitoring of cytosolic calcium reveals subtypes of rat lactotrophs with distinct responses to dopamine and thyrotropin-releasing hormone.

The cytosolic free calcium concentration, [Ca2+]i, was monitored in single rat lactotrophs in primary culture with the fluorescent probe Fura 2. It was found that lactotrophs are very heterogeneous in their [Ca2+]i response to TRH and dopamine, the major physiological regulators of PRL secretion. While in most lactotrophs TRH raises [Ca2+]i, the kinetics of this rise and the magnitude of its first and second phases vary considerably. For dopamine two clearly divergent response types can be observed. In part of the lactotrophs dopamine causes a lowering of [Ca2+]i from elevated levels, whereas in about 40% of the lactotrophs dopamine leads to a transient rise of [Ca2+]i. The present study reveals subclasses of lactotrophs with distinct [Ca2+]i response characteristics. It is suggested that such response type heterogeneity is a means of optimizing the secretory response to the complex regulatory influences on the pituitary.

Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials.

Electrical activity in non-neuronal cells can be induced by altering the membrane potential and eliciting action potentials. For example, hormones, nutrients and neurotransmitters act on excitable endocrine cells. In an attempt to correlate such electrical activity with regulation of cell activation, we report here direct measurements of cytosolic free Ca2+ changes coincident with action potentials. This was achieved by the powerful and novel combination of two complex techniques, the patch clamp and microfluorimetry using fura 2 methodology. Changes in intracellular calcium concentration were monitored in single cells of the pituitary line GH3B6. We show that a single action potential leads to a marked transient increase in cytosolic free calcium. The size of these short-lived maxima is sufficient to evoke secretory activity. The striking kinetic features of these transients enabled us to identify oscillations in intracellular calcium concentration in unperturbed cells resulting from spontaneous action potentials, and hence provide an explanation for basal secretory activity. Somatostatin, an inhibitor of pituitary function, abolishes the spontaneous spiking of free cytosolic Ca2+ which may explain its inhibitory effect on basal prolactin secretion. Our data therefore demonstrate that electrical activity can stimulate Ca2+-dependent functions in excitable non-neuronal cells.

Hyperalgesia in spontaneous and experimental animal models of diabetic neuropathy.

Hyperactivity of nociceptive C-fibers has been recently described in diabetic BB/Wistar rats. This study assesses the association of hyperalgesia, using an analgesy-meter, with elevated glycosylated haemoglobin levels in three animal models of diabetic and nutritional neuropathies: Psammomys obesus (sand rat), streptozotocin-treated and galactose-fed rats. Pain threshold measurements (paw pressure test) and motor nerve conduction velocities were recorded in controls (n = 75), hyperinsulinaemic (n = 16), insulin-deficient (n = 46) und galactosaemic (n = 12) animals. The reproducibility of the paw pressure test, evaluated by a correlation coefficient, was statistically significant (p less than 0.001). When compared with their controls (396 +/- 18 g), the average pain threshold in young diabetic sand rats (309 +/- 17 g) was found to be markedly reduced and to correlate inversely (p less than 0.001) with their respective HbA1c levels (mean 4.9 versus 7.4%). Acute, subacute and chronic streptozotocin-diabetic rats displayed a reduction of pain threshold (p less than 0.001) associated with slowed motor nerve conduction velocities (p less than 0.001). Similarly, galactose-feeding over 4 weeks resulted in an elevation of glycosylated haemoglobin levels with significant (p less than 0.001) reductions of pain threshold and motor nerve conduction velocity. It is concluded that hyperalgesia is a constant feature of sensory dysfunction in spontaneous and experimental models of diabetic neuropathy.

Current aspects of research on the pathogenesis of diabetic neuropathy.

Diabetic neuropathy contributes to the long-term complications of diabetes mellitus. Its pathogenesis is still unknown although a host of abnormalities in peripheral nerves in human diabetics and animal models has been discovered. This article emphasizes the current metabolic hypotheses: increased polyol pathway, decreased myoinositol nerve content; altered phospholipid metabolism, decreased Na+/K+ ATPase activity and non-enzymatic glycosylation of proteins. Future perspectives relevant to animal and clinical research and new therapeutic aspects are also discussed.

Pertussis toxin selectively abolishes hormone induced lowering of cytosolic calcium in GH3 cells.

Pertussis toxin, PT, abolishes inhibitory regulation of adenylate cyclase by cell surface receptors. Inhibitors of adenylate cyclase in GH3 cells, namely somatostatin and the muscarinic cholinergic agonist carbachol, lower the cytosolic free Ca2+ concentration. [Ca2+]i and cause hyperpolarization. These responses are selectively abolished by PT. It is concluded that the effects of somatostatin and carbachol to lower [Ca2+]i and to hyperpolarize are secondary to their inhibitory action on adenylate cyclase. In contrast, PT does not impair the TRH induced rise in [Ca2+]i in GH3 cells demonstrating that the coupling of TRH receptors to Ca2+ mobilization is not mediated by a PT substrate.

Lowering of cytosolic free Ca2+ by carbachol, a muscarinic cholinergic agonist, in clonal pituitary cells (GH3 cells).

Muscarinic cholinergic agonists have been shown to inhibit PRL secretion in normal and tumor-derived pituitary cells. Evidence from experiments with the fluorescent Ca2+ probe quin 2 shows that carbachol, acting through muscarinic acetylcholine receptors, lowers the cytosolic free Ca2+ concentration ([Ca2+]i), in GH3 cells. A decrease in [Ca2+]i is observed rapidly after carbachol addition, the lowered steady state [Ca2+]i is maintained, and upon the addition of atropine [Ca2+]i returns to the initial basal value. The lowering from a basal [Ca2+]i, averaging 110 +/- 2 nM (+/- SEM, n = 9), to a steady state [Ca2+]i of 63 +/- 4 nM (+/- SEM, n = 5) at 10 micron carbachol is dose dependent, a significant decrease from basal [Ca2+]i being observed at 0.1 micron. Carbachol does not prevent TRH-induced mobilization of Ca2+ but attenuates the resulting rise in [Ca2+]i. The lowering of steady state [Ca2+]i and the attenuation of the rise in [Ca2+]i provoked by stimulators of PRL secretion could explain the inhibition of both basal and stimulated PRL secretion. Concomitantly with the action on [Ca2+]i, carbachol causes hyperpolarization of GH3 cells. Together with the established inhibition of adenylate cyclase by muscarinic cholinergic agonists, these findings suggest a relation between changes in trans-membrane Ca2+ fluxes and cAMP generation.

Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells).

Changes in the cytosolic free Ca2+ concentration, [Ca2+]i, have been proposed to mediate the regulation of the secretion of pituitary hormones by hypothalamic peptides. Using an intracellularly trapped fluorescent Ca2+ probe, quin2, [Ca2+]i was monitored in GH3 cells. Somatostatin lowers [Ca2+]i in a dose dependent manner from a prestimulatory level of 120 +/- 4 nM (SEM, n = 13) to 78 +/- 9 nM (n = 5) at 10(-7)M; the effect is half maximal at 2 X 10(-9) M somatostatin. The decrease in [Ca2+]i occurs rapidly after somatostatin addition and a lowered steady state [Ca2+]i is maintained for several minutes. Somatostatin does not inhibit the rapid rise in [Ca2+]i elicited by thyrotropin releasing hormone (TRH) and can still cause a decrease in [Ca2+]i in the presence of TRH (10(-7)M). Concomitantly with its action on [Ca2+]i somatostatin causes hyperpolarization of GH3 cells assessed with the fluorescent probe bis-oxonol. The lowering of [Ca2+]i by somatostatin is however not only due to reduced Ca2+ influx through voltage dependent Ca2+ channels, since it persists in the presence of the channel blocker verapamil. These results suggest that somatostatin may exert its inhibitory action on pituitary hormone secretion by decreasing [Ca2+]i.

Polyphosphoinositide hydrolysis by phospholipase C is accelerated by thyrotropin releasing hormone (TRH) in clonal rat pituitary cells (GH3 cells).

Thyrotropin releasing hormone (TRH) accelerates the turnover of phosphatidylinositol in GH3 cells ('phospholipid response'). From the analysis of inositol phosphates in the presence of Li+ which inhibits their dephosphorylation, it can be concluded that the hydrolysis of phosphatidylinositol 4,5-biphosphate, and possibly of phosphatidylinositol 4-phosphate by phospholipase C is markedly accelerated by TRH. It appears that this reaction initiates the acceleration of phosphatidylinositol turnover. The specificity of hormonally regulated phospholipase C reaction for polyphosphoinositides has important implications for the potential role of the phospholipid response as a mechanism of membrane signal transduction.

The application of HPLC methodology for the analysis of receptor mediated changes in phosphoinositide metabolism.

HPLC methodology has found wide application in analytical problems in biochemistry. To study the metabolism of phosphatidylinositol and its regulation by receptor mediated events, HPLC could be a valuable technique. It has been recently demonstrated that a variety of hormones and neurotransmittors act to stimulate hydrolysis of phosphoinositides by a phospholipase C. To monitor this reaction, we have analysed the formation of radiolabelled inositol phosphates from phosphoinositides. The present paper describes a rapid HPLC procedure, to separate inositol phosphates from myo-inositol, which could be used in pharmacological studies of receptors linked to phosphoinositide hydrolysis. The potential of the application of HPLC to the analysis of the phospholipids involved is discussed.

Peptides of the neurointermediary lobe of rat pituitary stimulate GH secretion in vitro.

alpha-MSH and other fragments of ACTH are potent stimulators of GH release in vivo. The action of such peptides and of extracts of the neurointermediary lobe (NIL) of rat pituitary, a source of endogenous MSH-related peptides, on GH release was investigated in vitro. Peptides with the core sequence of alpha-MSH stimulate GH secretion by primary cultures of rat anterior pituitary cells; however, both the absolute and the relative potencies of these peptides exclude the involvement of melanotropic receptors comparable in specificity to the extrapituitary receptors for these hormones. Extracts of the NIL of rat pituitary stimulate GH release in vitro and the bulk of the releasing activity can be attributed to one (or several) factors with an apparent mass of approx. 10 000 M.W. that can be partially purified by HPLC. The active principle appears to be distinct from both beta-LPH and the human pancreatic GHRF. Thus, while rat NIL contains GH-releasing activity that can be demonstrated in vitro, a direct link to the potent action of MSH-related peptides on GH release in vivo cannot be established, and the action of these peptides in vivo must therefore rely on mechanisms which are not expressed in the in vitro system.