High-density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells

Hepatic NK cells are more cytotoxic than blood NK cells against tumor cells. To understand the basis of this difference in cytotoxicity we analyzed RNA derived from freshly isolated rat blood and hepatic NK cells [high-density (HD) and low-density subpopulations] by high-density oligonucleotide arrays (Affymetrix), containing about 9,000 genes and expressed sequence tags. IL-2-treated blood NK (A-NK) cells and IL-2-treated hepatic HD cells were used as a reference of NK cell activation. About 150 genes and expressed sequence tags were differentially expressed between hepatic and blood NK cells. Surprisingly, more than half of the increased expressed genes in hepatic NK cells were not increased in A-NK cells. Differentially expressed genes like the stem cell factor receptor c-kit and the chemokine receptor CCR5 can contribute to the homing and differentiation of hepatic NK cells in the liver sinusoids. Several of the differentially expressed genes can possibly contribute to the enhanced cytotoxic activity of hepatic NK cells: cell membrane receptors like NKG2D, NKG2C, CD94, ecto-ATPase; signaling molecules like phosphatidylinositol 3-kinase; granule-associated effector molecules like granzymes and defensin NP3. Moreover, it appears that redirection of cytotoxic granules and increase in intracellular Ca2+ are convergence points of several of these genes.     

Mechanisms underlying changes in functional properties of red cells in acute action of carbon monoxide

It has been experimentally established that a single impact of carbon mono-oxide in concentration 4000 mg/m3 during 75 minutes (CL50) in rats is accompanied with disturbance of functional activities of blood red cells in the form of a decrease of deformability and an increase of aggregative capacity of erythrocytes which are most evident to the end of the first day after carbon mono-oxide administration. These changes are combined with structural modification and changes in metabolism of erythrocytic membrane. The signs of structural modification of erythrocytic membrane in rats exposed to acute effect of carbon mono-oxide in a mean lethal concentration stay long remaining during elimination of carboxyhemoglobinemia (up to 14-21 days).     

Estimation of single channel conductance underlying synaptic transmission between pyramidal cells in the visual cortex

Axon collaterals originating from pyramidal cells are one of the most abundant presynaptic elements in the neocortical circuits. To understand a quantitative aspect of synaptic transmission between pyramidal cells, we attempted to estimate single channel conductance by applying non-stationary noise analysis to unitary excitatory postsynaptic currents. Simultaneous recordings were carried out in two pyramidal cells of superficial layers in visual cortical slices. Unitary postsynaptic currents, which were evoked by action potentials of presynaptic cells impaled with conventional sharp electrodes, were recorded from postsynaptic cells with whole-cell patch clamp techniques. Estimated single channel conductance was 12.8 3.8(S.D.) pS for kittens and 10.4 +/- 1.5 pS for rats. Dividing these values by the conductance for unitary postsynaptic currents, we calculated the number of non-N-methyl-D-aspartate receptor channels activated during the postsynaptic currents. The obtained estimates were 52 (kittens) and 41 (rats). To further estimate the number of channels involved in each quantal event, we analysed amplitude histograms of miniature and spike-evoked excitatory postsynaptic currents. The derived number of estimates from these two kinds of histograms agreed quite well; about 20 channels were required for individual quantal events. Assuming open probability of non-N-methyl-D-aspartate receptor channels to be 0.7, our results suggest that the number of channels available for synaptic transmission between individual pyramidal cells would be 74 (kittens) and 59 (rats). We propose that at pyramidal-pyramidal synapses, the number of open channels is several times smaller than that previously reported for the synapses between geniculo-cortical afferent and layer IV spiny stellate cells.     

Pre- and postnatal differences in membrane, action potential, and ion channel properties of rostral nucleus of the solitary tract neurons

There is little known about the prenatal development of the rostral nucleus of the solitary tract (rNST) neurons in rodents or the factors that influence circuit formation. With morphological and electrophysiological techniques in vitro, we investigated differences in the biophysical properties of rNST neurons in pre- and postnatal rats from embryonic day 14 (E14) through postnatal day 20. Developmental changes in passive membrane and action potential (AP) properties and the emergence and maturation of ion channels important in neuron function were characterized. Morphological maturation of rNST neurons parallels changes in passive membrane properties. Mean soma size, dendritic branch points, neurite endings, and neurite length all increase prenatally. whereas neuron resting membrane potential, input resistance, and time constant decrease. Dendritic spines, on the other hand, develop after birth. AP discharge patterns alter in pre- and postnatal stages. At E14, neurons generated a single TTX-sensitive, voltage-gated Na(+) AP when depolarized; a higher discharge rate appeared at older stages. AP amplitude, half-width, and rise and fall times all change during development. Responses to current injection revealed a number of voltage-gated conductances in embryonic rNST, including a hyperpolarization-activated inward current and a low-threshold Ca(2+) current that initiated Ca(2+) spikes. A hyperpolarization-activated, transient outward potassium current was also present in the developing neurons. Although the properties of these channels change during development, they are present before synapses form and therefore, can contribute to initial establishment of neural circuits, as well as to the changing electrophysiological properties in developing rNST neurons.     

DRG慢性痛信号产生的细胞兴奋性分型及离子通道机制

目的:研究Hodgkin提出的兴奋性分型是否存在于背根神经节(Dorsal Root Ganglion,DRG),并对该分型及介导该分型的离子通道电流的变化在慢性痛信号产生中的重要作用进行探讨。方法:本研究通过制备背根节慢性压迫模型(chronic compression of DRG,CCD),在整节消化后的DRG大中细胞进行全细胞膜片钳记录。结果:依照神经元受刺激后产生动作电位的特征,79只大鼠的311例神经元可分为三种类型。在正常对照组(n=179),1、2、3型神经元所占比例分别为13%、27%和60%;在CCD组(n=134)比例则分别为27%、33%和40%。统计分析显示慢性压迫损伤后,1型神经元分型比例明显增加,而3型神经元分型比例显著减少(P0.05)。正常DRG神经元中,持续钠电流(INaP)的幅值在1、2型神经元中比3型神经元明显要高(P0.05)。CCD术后INaP在1、2型神经元中均增加,但仅在1型神经元有显著性差异(P0.05)。结论:DRG大中细胞在慢性压迫损伤后,神经元兴奋性分型中1、2型神经元的比例增多和1、2型神经元上INaP电流的增加,共同造成了由DRG向更高级中枢的信息传递的加强。     

Kinetic analysis of K+ ion channel function in lymphokine-activated killer (LAK) cells

K+ ion channels of lymphocytes have been implicated in cellular differentiation, activation and cytolytic functions. We previously demonstrated that K+ channel blockers modulate lytic activity of CTLs and LAK cells. In the present study, we define and quantitate the inhibitory effects of ion channel blockers on the lytic process using kinetic analysis of lysis. The K+ channel blocker, 4-aminopyridine, the neuroendocrine monoamine, serotonin, its agonist, quipazine, and the Ca++ dependent K+ channel blocker, quinidine were found to non-competitively inhibit the lytic process in a dose-dependent manner. These compounds inhibit lytic activity by causing a decrease in the maximum velocity (Vmax) by which LAK cells lyse tumor targets. These ion channel blockers did not alter effector or target cell viability or the binding of LAK cells to tumor cells. The inhibitory effects occurred at the effector cell level, since preincubation of LAK effector cells resulted in a dose-dependent decrease in Vmax which was related to a slower rate of target cell lytic programming (k2) by the LAK effector cells. Modulation of LAK cell lytic function occurs at a post-binding step, perhaps in the generation or release of the lytic signal.     

Texture analysis of protein distribution images to find differences due to aging and superfusion

Many pathologies are age-related,e.g., cardiovascular disease generally occurs in midlife and cancer later in life. This suggests that aging predisposes the body to pathology. Plasma protein spatial distribution images of rat mesentery extracellular matrix (ECM) show texture due to the ECM structures, and there is an age-related decrease in tissue protein that may be related to matrix structure changes. The objective of this study was to compare changes in protein image texture under two conditions: superfusion with normal saline solution and aging. The decrease in soluble protein concentration during superfusion is a washout process, while the mechanism for the age-related decrease in tissue protein is unknown. Therefore, effects of aging and superfusion on tissue protein image texture were compared. Spatial co-occurence matrix and Fourier analysis techniques have been used for texture evaluation. Superfused images showed a more uniform protein texture. There were gradual age-related changes in image texture parameters. Entropy increased with age from 140 to 630 days, indicating that protein distribution became more disorganized. The results suggest that changes in protein image texture are due to age-related alterations in matrix structure because removing only protein by superfusion had opposite effects on texture parameters.     

Increase in deaths caused by HIV infection due to changes in rules for selecting underlying cause of death

With implementation of the (ICD-10), for U.S. vital statistics in 1999, the criteria for selecting HIV infection as the underlying cause of death were expanded. To estimate the effect of ICD-10 rules on the number of deaths attributed to HIV infection, we applied a simplified version of ICD-10 rules to data on causes of death from all U.S. death certificates for 1998 (previously classified by rules of the 9th revision of ICD [ICD-9]) and calculated the resulting increase in deaths for which HIV infection was selected as the underlying cause. Of the 17,186 deaths in 1998 with any mention of HIV infection on the death certificate, ICD-10 rules selected HIV infection as the underlying cause for 15,145, which was 1,719 (13%) more than the 13,426 for which it had been selected by ICD-9 rules. The proportional increase differed by demographic group, being less among non-Hispanic blacks (9%) and Hispanics (13%) than among non-Hispanic whites (18%). Thus, comparison of deaths attributed to HIV infection in 1999 or later with those in 1998 or earlier should take into account the changes in ICD rules for selecting the underlying cause of death.     

Sensitivity to immunodepressant action of cyclophosphamide: analysis of interstrain differences in mice

In one of our previous studies (Pevnitsky et al., Bull. exp. Biol. Med., 83, 438-440, 1977), we have found significant differences between various strains of mice in the sensitivity to immunodepressant action of cyclophosphamide (CP). The degree of these differences was not determined by the level of their immune response which indicates that the cause of the interstrain differences lies in a specific reaction of mice to the immunodepressant. The main parameters of CP effect which may be responsible for variable sensitivity to the immunodepressant action in vivo were studied in several murine strains (Balb/cJLacSto, CBA/CaLacSto, and DBA/2JSto): (1) rate of the preparation activation in liver microsomes; (2) pharmacokinetics of NBP-metabolites in the blood serum; (3) immunodepressant action of the in vivo activated CP; (4) sensitivity of immunocompetent target cells to activated CP effect. It was found that DBA/2 mice are the most sensitive to CP in vivo. The level of "active" CP in their blood serum is higher than in BALB/c mice. Besides, they are characterized by a higher sensitivity of immunocompetent cells compared to BALB/c and CBA mice.     

The roles of leukaemic and residual normal cells in the proliferative response of chronic lymphocytic leukaemic lymphocytes to mitogens.

The proliferative response of lymphocytes from patients with chronic lymphocytic leukaemia (CLL) to the polyclonal activators, phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) correlates inversely with the logarithm of the circulating lymphocyte concentration in vivo (P less than 0.001). This suggests that the response is due primarily to residual normal cells in the circulation. In support of this postulate, dilution of normal cells reproduced the effect and also induced a delayed response to PHA, which is found frequently with CLL lymphocytes. A combination of autoradiography and immunofluorescence microscopy identified both B and T cells in the responding population in similar proportions from both normal and CLL donors. These data demonstrate that the 3H-thymidine assay used in most mitogenic studies is not suitable alone for investigating the functional capacity of leukaemic lymphocytes in CLL and other diseases involving a gross perturbation of lymphocyte populations.     

Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specific differences

The diagnosis of prion diseases, such as scrapie and BSE, has traditionally relied upon the identification of the disease-associated form of the prion protein, PrP(Sc), based on its resistance to digestion by proteinase K (PK). A more recent development is the conformation-dependent immunoassay (CDI), which distinguishes between PrP Sc and normal PrP (PrP C) based on their differing solubility in guanidine hydrochloride rather than resistance or sensitivity to PK. We have developed a CDI-formatted sandwich immunoassay for the measurement of PrP Sc in sheep brain, which discriminates between clinically affected scrapie cases (natural or experimental) and uninfected controls of the same PrP genotype. Using this method, we have shown for the first time that, in sheep, the PrP genotype has a significant influence on the amount of PrP Sc deposited in the brains of animals experimentally infected with scrapie.     

Large-scale methylation analysis of human genomic DNA reveals tissue-specific differences between the methylation profiles of genes and pseudogenes

Cytosine in CpG dinucleotides is frequently found to be methylated in the DNA of higher eukaryotes and differential methylation has been proposed to be a key element in the organization of gene expression in man. To address this question systematically, we used bisulfite genomic sequencing to study the methylation patterns of three X-linked genes and one autosomal pseudogene in two adult individuals and across nine different tissues. Two of the genes, SLC6A8 and MSSK1, are tissue-specifically expressed. CDM is expressed ubiquitously. The pseudogene, psi SLC6A8, is exclusively expressed in the testis. The promoter regions of the SLC6A8, MSSK1 and CDM genes were found to be essentially unmethylated in all tissues, regardless of their relative expression level. In contrast, the pseudogene psi SLC6A8 shows high methylation of the CpG islands in all somatic tissues but complete demethylation in testis. Methylation profiles in different tissues are similar in shape but not identical. The data for the two investigated individuals suggest that methylation profiles of individual genes are tissue specific. Taken together, our findings support a model in which the bodies of the genes are predominantly methylated and thus insulated from the interaction with DNA-binding proteins. Only unmethylated promoter regions are accessible for binding and interaction. Based on this model we propose to use DNA methylation studies in conjunction with large-scale sequencing approaches as a tool for the prediction of cis-acting genomic regions, for the identification of cryptic and potentially active CpG islands and for the preliminary distinction of genes and pseudogenes.     

Lectin cytochemistry reveals differences between hamster trachea and bronchus in the composition of epithelial surface glycoconjugates and in the response of secretory cells to neutrophil elastase

Hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate an abnormally high number of secretory granules in bronchial but not tracheal epithelial cells. We employed lectin cytochemistry to investigate possible differences in the epithelial cell surface glycoconjugate layer in trachea compared to bronchus which might explain the regional dissimilarity in response to HNE. Portions of glutaraldehyde-fixed trachea and bronchi were incubated in one of several ferritin-labeled lectins prior to embedding for transmission electron microscopy. Lectins from Ricinus communis, Helix pomatia, and Triticum vulgaris bound to the surface of tracheal secretory cells in moderate to profuse amounts, while most bronchial secretory cells showed little or no label with these lectins. Gold-labeled Helix pomatia agglutinin (HPA), a lectin specific for secretory cells, showed a decrease in surface binding to all tracheal secretory cell types within 2 h of HNE instillation, compared to saline controls. In contrast, the majority of bronchial secretory cells showed an HNE-induced increase in surface label from extremely low levels in saline controls. The low levels of lectin binding to bronchial cells, in contrast to the trachea, may indicate the lack of a protective surface glycoconjugate coat, thus explaining the vulnerability of these cells to HNE. The rise in number of accessible HPA binding sites on the surface of bronchial secretory cells exposed to HNE may represent an important event in the pathologic accumulation of secretory granules by these cells.     

Intracellular ion exchange between cytoplasmic potassium and granule histamine, an integrated link in the histamine release machinery of mast cells

Rat peritoneal mast cells isolated by gradient centrifugation in Percoll were placed between two membrane filters in a Sartorius filter apparatus and superfused with isotonic balanced salt solutions or with deionized isotonic sucrose. Histamine was released according to ion exchange kinetics. Our explanation of the observed phenomena is as follows. The superfusion induces a flow of cytoplasmic K+ ions across the histamine-containing granules, resulting in an ion exchange K+ in equilibrium Hi+ ions at the histamine binding sites. The concomitant equimolar outflow of histamine and potassium is considered to be due to a functional interplay between two histamine pools, a release and a donor pool. As the result of the K+ in equilibrium Hi+ ion exchange at the histamine binding sites of the release pool, these sites become transiently occupied by K+ ions only to be immediately reoccupied by Hi+ ions from the donor pool. The observed equimolar outflows are consistent with a 1/1 molar ratio in the exchange between histamine and potassium ions. The essential role of cytoplasmic potassium in the histamine release mechanism is a new and important observation with possible implications not only as to histamine release in general (including so-called 'spontaneous' histamine release) but also as to the release of biogenic amines and other positively charged substances stored in granules in ionic linkage to the matrix.     

Intracellular ion exchange between cytoplasmic potassium and granule histamine,an integrated link in the histamine release machinery of mast cells*

Rat peritoneal mast cells isolated by gradient centrifugation in Percoll were placed between two membrane filters in a Sartorius filter apparatus and superfused with isotonic balanced salt solutions or with deionized isotonic sucrose. Histamine was released according to ion exchange kinetics. Our explanation of the observed phenomena is as follows. The superfusion induces a flow of cytoplasmic K⁺ ions across the histamine-containing granules, resulting in an ion exchange K⁺←Hi⁺ ions at the histamine binding sites. The concomitant equimolar outflow of histamine and potassium is considered to be due to a functional interplay between two histamine pools, a release and a donor pool. As the result of the K⁺←SHi⁺ ion exchange at the histamine binding sites of the release pool, these sites become transiently occupied by K⁺ ions only to be immediately reoccupied by Hi⁺ ions from the donor pool. The observed equimolar outflows are consistent with a 1/1 molar ratio in the exchange between histamine and potassium ions. The essential role of cytoplasmic potassium in the histamine release mechanism is a new and important observation with possible implications not only as to histamine release in general (including so-called ‘spontaneous’ histamine release) but also as to the release of biogenic amines and other positively charged substances stored in granules in ionic linkage to the matrix.     

Scanning EM of resting gastric parietal cells reveals a network of cytoplasmic tubules and cisternae connected to the intracellular canaliculus

Using a recently developed fixation technique for parietal cells (Sugai et al., Acta Anat Nippon 1995:70:S79, 1999:74:S101), we have reinvestigated the organization of the cytoplasmic membrane system in the resting stomach by ultra-high-resolution scanning electron microscopy (SEM). Rat gastric mucosae were microwave-fixed in cacodylate buffer [334 milliosmoles/kg H(2)O (mOsm)], to which 1.0% glutaraldehyde and 0.5% formaldehyde were added. Specimens examined by transmission electron microscopy (TEM) of thin sections revealed cytoplasm packed with tubular membranes similar to images detected by rapid-freeze/freeze-substitution fixation which is generally considered to cause minimal structural alterations. To render the cytoplasmic membranes visible by SEM, fixed mucosae were frozen, fractured, and the exposed cytoplasm of parietal cells was macerated by the aldehyde-osmium-DMSO-osmium procedure. With much of the cell matrix and filaments removed, SEM revealed numerous 30-60 nm tubules which formed a meshwork and also small cisternae. The cytoplasmic surface of the tubules was smooth while some cisternal areas had attached polyribosomes. Vesicles or isolated tubules were not found in appropriately macerated parietal cells. The cytoplasmic surface of the intracellular canaliculus was smooth except for round openings representing the bases of macerated microvilli. In favorable sites connections of the tubular membranes to the canaliculi were clearly visible. Stereo pair views were particularly useful to demonstrate these continuities. Connections between these two membrane compartments suggest the probability of rapid membrane transposition.     

Histomorphological study of cellular interactions between stromal and haemopoietic stem cells in normal and leukaemic bone marrow

The interactions between haemopoietic and stromal elements are crucial for stem cell proliferation and differentiation. The bulk of this evidence is derived from experiments in rodents and in-vitro culture studies. We have studied the spatial relationships between the stromal and haemopoietic components and their cellular composition in histological sections of bone marrow (BM) from seven healthy fetuses, 10 normal adults and over 60 patients with acute myeloid leukaemia (AML) and chronic granulocytic leukaemia (CGL) at different stages of the disease. During the early developmental stage (16–18 weeks) fetal BM showed focal haemopoiesis with a characteristic spatial localization of haemopoiesis near bony trabeculae and around small blood vessels. In AML following the treatment-induced hypoplasia, large uniform unilocular fat cells arranged in groups designated ‘structured fat’ developed from scattered multilocular precursor fat cells. Early foci of haemopoietic regeneration were present almost exclusively in areas of structured fat. In the marrow of patients with CGL in blast transformation (BT) treated by intensive therapy and autografting with cryopreserved haemopoietic stem cells, the haemopoiesis was focal. Clusters of regenerating erythroid precursors or of megakaryocytes were seen in intimate contact with marrow sinusoids and granulopoietic precursors in intimate association with small blood vessels and also in close contact with the endosteal surface of bony trabeculae. We conclude that the endosteal cells, fat cells and the vascular endothedial cells comprise the critical non-haemopoietic stromal elements of human BM. The close associations observed between the regenerating haemopoietic cells and the stromal cells provide strong evidence in support of the existence of a permissive haemopoietic micro-environment in man and emphasize the structural and functional interrelationships that exists between bone, fat, the microvascular system and haemopoiesis in human bone marrow.