Horseradish peroxidase as a label of injured cells

Synopsis The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H₂O₂ medium for the shortest possible period.Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.     

Transport systems of serine at the brain barriers and in brain parenchymal cells

D-Serine is a co-agonist for NMDA-type glutamate receptors. Although D-serine levels in CSF and interstitial fluid (ISF) affect CNS function, the regulatory system remains to be fully understood. Therefore, the purpose of this study was to investigate d-serine transport across the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) and in brain parenchymal cells. D-Serine microinjected into the cerebrum was not eliminated, suggesting a negligible contribution of D-serine efflux transport at the BBB. In contrast, D-serine was taken up from the circulating blood across the BBB via a carrier-mediated process. D-Serine elimination clearance from CSF was fourfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. The characteristics of D-serine uptake by isolated choroid plexus were consistent with those of Na(+)-independent alanine-serine-cysteine transporter 1 (asc-1). Uptake of D-serine by brain slices appeared to occur predominantly via asc-1 and Na(+)-dependent alanine-serine-cysteine transporter 2. These findings suggest that the regulatory system of D-serine levels in ISF and CSF involves (i) asc-1 at the BCSFB, acting as a major pathway of D-serine elimination from the CSF, (ii) blood-to-brain and blood-to-CSF influx transport of D-serine across the BBB and BCSFB, and (iii) concentrative uptake of D-serine by brain parenchymal cells.     

Horseradish peroxidase uptake and crinophagy in insulin-secreting cells

Upon exposure of pancreatic B cells to exogenous horseradish peroxidase (HRP), a population of secretory granules becomes HRP-labelled. In isolated islets of Langerhans, we studied the fate of HRP-labelled secretory granules during a pulse-chase experiment with HRP in order to assess their relationship with lysosomes containing secretory granule cores. These structures (crinophagic or multigranular bodies) were previously shown to be a site of insulin degradation (Orci et al., J cell biol 98 (1984) 222) [4]. After a 15-min pulse of peroxidase, the number and volume density of HRP-labelled secretory granules decreased over an 85-min chase period, during which the number and volume density of multigranular bodies labelled with HRP was significantly increased. At both time points, the surface density of HRP-labelled Golgi elements was very small compared with that of unlabelled ones. By autoradiography after a 5-min pulse of [3H]leucine and a 55-min chase, followed by a 15-min pulse of HRP and a 85-min chase, we could show that the majority of HRP-containing secretory granules were not radioactively labelled granules. These results suggest that: The low degree of HRP labelling of the Golgi makes it unlikely that secretory granules derive their HRP by budding from HRP-labelled cisternae. HRP-labelled SGs are preferentially transferred to MGBs (which become HRP-labelled) for prospective degradation. HRP labelling does not involve newly-formed mature secretory granules.     

EEG and implanted sources in the brain

Localisation procedures are based on models of the EEG that are relatively simple. The models are based on assumptions and choices of parameters that can be mistaken. Thus, it is crucial to validate the localisation procedures used in EEG. One of the options is to use the data obtained with electrodes that are implanted within the brain of an epileptic patient as part of the pre-surgical evaluation. When one of two neighbouring electrodes is used as a current source and the other as a current sink this can be regarded as a current dipole. The current injected has to be below the threshold for activation of cells. The position of this dipole can be deduced from magnetic resonance or X-ray images. The current dipole gives rise to a potential distribution at the scalp that can be measured by EEG. The measurements can be compared with the potential distribution that is calculated in a forward computation. Another method is to use the measured potential at the scalp to localize the source and to compare the result with the actual position of the dipole. In this paper the measured potential distributions at the scalp due to implanted dipoles were used to evaluate different volume conductor models. Since intracerebral and subdural electrodes were introduced through trephine holes over the fronto-central areas, and the diameter of the holes was rather large, approximately 23 mm, special effort was put into modelling the skull. Two important assumptions could be validated in this study: the electric currents within the head are Ohmic and a dipole can be used to model the induced electric activity of pairs of contacts on subdural electrodes or intra cerebral electrodes.     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.     

Corticosteroid receptors in the brain as signal systems of stress and adaptation

The modern literature date and own authors investigation allow to consider brain corticosteroid receptors as the importance system of stress and adaptation. The main attention is paid for functional meaning and distribution of corticosteroid receptors in brain regions. The role of brain corticosteroid receptors in transduction of hormonal signal and its modification different regulatory factors are discussed.     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.Key words: induced pluripotent stem cells (iPSCs), blood, B lymphocytes, hematopoietic differentiation, hepatic differentiation, disease modeling, drug testing     

Amino acid uptake systems in lizard and chick brain cells

The uptake of seven amino acids, -aminoisobutyric acid (AIB), cyclo-leucine (cyclo-Leu), -aminobutyric acid (GABA), glycine (Gly), glutamic acid (Glu), lysine (Lys), and taurine (Tau), representatives of different amino acid transport systems, was studied in slices of brain from Tokay lizards and White Leghorn chicks. In descending order, the rate of the initial uptake of the amino acids in both species was Glu>Gly>GABA>Cyclo-Leu>AIB>Lys>Tau. The substrate specificities and the differences in sodium and temperature dependence of the uptake of the amino acids indicate the presence of several distinct amino acid transport systems, some sodium-dependent and some sodium-independent. The structural specificity of amino acid transport classes in the brain of these species is similar to that in other vertebrate brain preparations.Special issue dedicated to Dr. Santiago Grisolia.     

Hairy root type plant in vitro systems as sources of bioactive substances

“Hairy root” systems, obtained by transforming plant tissues with the “natural genetic engineer” Agrobacterium rhizogenes, have been known for more than three decades. To date, hairy root cultures have been obtained from more than 100 plant species, including several endangered medicinal plants, affording opportunities to produce important phytochemicals and proteins in eco-friendly conditions. Diverse strategies can be applied to improve the yields of desired metabolites and to produce recombinant proteins. Furthermore, recent advances in bioreactor design and construction allow hairy root-based technologies to be scaled up while maintaining their biosynthetic potential. This review highlights recent progress in the field and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.     

Distribution of horseradish peroxidase-labeled neurons giving rise to descending fiber systems in the basal ganglia and hypothalamus in cats

The distribution of neurons giving rise to various descending fiber systems to brain-stem structures in the basal ganglia (including amygdaloid nuclei) and hypothalamus of the cat was studied by the retrograde axonal transport of horseradish peroxidase method. Neurons in the medial part of the central nucleus and of the magnocellular part of the basal nucleus of the amygdaloid group were shown to send axons to the dorsal hippocampus, substantia nigra, lateral part of the central gray matter, and the mesencephalalic reticular formation and also to the region of the locus coeruleus and the lateral medullary reticular formation at the level of the inferior olives. The predominant source of projections to the hypothalamus and brainstem structures is the central amygdaloid nucleus, which also sends projections to the nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus nerve, and the superior cervical segments of the spinal cord. Uncrossed fiber systems descending from the basal ganglia terminate at the level of the pons, whereas uncrossed and crossed fiber systems descending from the dorsal and ventromedial hypothalamus can be traced into the spinal cord. The possible role of nuclei of the amygdaloid group, the hypothalamus, and their efferent projections in the regulation of somatic and vegetative functions and also of complex behavioral reactions is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 14–23, January–February, 1981.     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Horseradish peroxidase,lactoperoxidase, and ferritin as markers

Summary The occurrence of endocytotic mechanisms in human small intestinal absorptive cells was investigated by culturing biopsy specimens in the presence of horseradish peroxidase (HRP), lactoperoxidase (LPO), and ferritin. The results indicate that both HRP and LPO entered the cells by apical endocytosis, after which they were transported via apical vesicles and tubules to the lysosome-like bodies. Ferritin, which showed a distinct affinity for the cell-coat glycoproteins, was not interiorized by the absorptive cells.These findings suggest that although human absorptive cells have an endocytotic mechanism, possibly fluid-phase endocytosis, cell-coat glycoproteins are not taken up by the cells, as indicated by the absence of ferritin in the apical vesicles and tubules, as well as the lysosome-like bodies. These findings provide indirect support for our hypothesis that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport via a crinophagic mechanism (fusion of apical vesicles and tubules with lysosome-like bodies) rather than via an exocytotic-endocytotic mechanism.     

Role of the GABA-ergic systems of the brain in manifesting the activating effect of diazepam]

It was shown in experiments on rats that the selective blocker of GABA receptors bicuculline (2 mg/kg) does not decrease the activating effect of diazepam as to the reaction of self-stimulation. The GABA-mimetic muscimol (0.5 and 1 mg/kg) had no effect on self-stimulation rate, while in the dose of 2 mg/kg causing behavioral changes produced a powerful decrease in it (by 93.3%). During the combined administration of diazepam and muscimol (1 mg/kg and 0.5 mg/kg, respectively) no potentiation of diazepam effect was observed. It is suggested that diazepam-induced facilitation of the reaction of self-stimulation is not due to the alteration in the activity of GABA-ergic processes.     

Gibb's free energy as a index of the assimilation of carbohydrate sources by bacteria]

The author presents substantiation of the use of thermodynamic function--of free Gibbs' energy (--deltaG) for energy characteristics of enterobacteria periodic cultures on synthetic media. Examination of strains of E. coli K12P4X Hfr, E. coli PA-309, and Sh. newcastle 762S showed the --detlaG = f (N) curves to characterise the energy value of various carbohydrate sources more completely, and also permitted the choice of their optimal concentrations in synthetic media for each of the enterobacteria strain.