Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice. We have established transgenic mice carrying the anti-sense DNA to the gene encoding beta chain of the class II major histocompatibility complex (I-A) molecule. The amount of I-A molecule on splenic B lymphocytes from the mice was reduced in the presence of a large amount of the exogenous anti-sense RNA. The amount of I-A beta chain RNA was selectively reduced and inversely correlated with the amount of anti-sense RNA in the spleens. These results suggest that the I-A beta chain RNA is rapidly degraded by duplex formation with the anti-sense RNA in splenic B cells from the transgenic mice.


Evidence for elevation of cytochrome P4502E1 (alcohol-inducible form) mRNA levels in rat kidney following pyridine administration. The effects of pyridine on renal cytochrome P4502E1 (CYP2E1) expression in rat have been examined by immunoblot and Northern blot analyses. Immunoblot analyses revealed that 2E1 protein levels were elevated from 1.4- to 4.6-fold following pyridine administration in a dose- and time-dependent manner. Northern blot analyses revealed that renal 2E1 poly(A)+ RNA levels increased from 1.4- to 3.8-fold following pyridine treatment and that these increases in 2E1 mRNA paralleled the dose- and time-dependent increases in 2E1 protein content. In contrast, hepatic 2E1 poly(A)+ RNA levels failed to increase following these same dosing regimens, suggesting that metabolic alterations, such as those associated with starvation, were not etiologic factors in renal 2E1 induction. These results show that pyridine induced CYP2E1 in kidney and that elevation of renal 2E1 protein levels accompanying pyridine administration occurred at least partly as a consequence of increased 2E1 poly(A)+ RNA levels. The results of this research reveal that regulatory mechanisms governing CYP2E1 expression may differ in hepatic and renal tissues.


Lipopolysaccharide and cytokines induce a macrophage-type of nitric oxide synthase in bovine retinal pigmented epithelial cells. The present study demonstrates that bovine retinal pigmented epithelial cells, which are neuroectodermal in origin, produce nitric oxide (NO) upon treatment with interferon-gamma in the presence of lipopolysaccharide or tumor necrosis factor-alpha. NO production was measured by the accumulation of the stable endproduct NO2-. The biosynthesis of NO requires an induction period of approximately 12 hours and continues for at least 96 hours. The synthesis was abolished by the stereoselective inhibitors of NO synthase, NG-monomethyl-L-arginine and NG-nitro-L-arginine-benzylester. Cycloheximide and dexamethasone blocked cytokine-induced NO production. The results indicate that endotoxin and cytokines are capable of inducing NO synthase of the macrophage type, in retinal pigmented epithelial cells.


Identification of osteopontin in isolated rabbit osteoclasts. Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.


Rat copper/zinc superoxide dismutase gene: isolation, characterization, and species comparison. A 13 kb rat Cu/ZnSOD genomic clone has been purified from a rat liver genomic library and completely characterized by restriction mapping, detailed sequencing and Southern blot analysis. This gene spans approximately 6 kb and contains five exons and four introns. Comparison of rat, mouse, and human Cu/ZnSOD genes reveals a high conservation in genomic organization and exon-intron junctions, including an unusual 5'GC donor sequence at the first intron. The gene contains a TATA box as well as an inverted CCAAT box, a feature common to both the mouse and human genes. Furthermore, several repeats were identified in the 5' promoter region of this gene, and these regulatory elements are also strikingly conserved in these three species.


Marked induction of hepatocyte growth factor mRNA in intact kidney and spleen in response to injury of distant organs. Hepatocyte growth factor (HGF) is a potent mitogen for various epithelial cells, including mature hepatocytes and renal tubular cells. Here, HGF mRNA was found to be markedly increased in non-injured kidney and spleen, when the liver or kidney in rats was injured by 70% partial hepatectomy or unilateral nephrectomy. HGF mRNA increased to 3-4 fold higher level than the normal in the kidney and spleen as well as in the remnant liver after partial hepatectomy. Similarly, HGF mRNA markedly increased in the spleen as well as in the remnant kidney after unilateral nephrectomy. These results suggest that the onset of injury to the liver or kidney may be recognized by distal non-injured organs by the signalling of a humoral factor and that HGF derived from these organs may be involved in the regeneration of liver or kidney, through an endocrine mechanism.


Effect of repeated oral doses of a novel immunosuppressive macrolide lactone on hepatic mixed-function oxidase system in the rat. Comparative study with ciclosporin. Effect of pretreatment of rats with FK506((-)-(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-17-allyl-1,14- dihydroxy-12-[(E)-2-[(1R,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1- methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4- azatricyclo-[22.3.1.0(4.9)]octacos-18-ene-2,3,10,16-tetrone hydrate, CAS 104987-11-3) on microsomal cytochrome P-450 system and oxidations of the administered drug and other model substrates were studied and compared with those of a pharmacologically related drug, ciclosporin (cyclosporin A). Oral treatment of male Sprague-Dawley rats with FK506 (0.4, 2 or 10 mg/kg/d) for 7 days did not decrease microsomal content of total cytochrome P-450 in livers, but rather increased the content in groups with the dose of 0.4 or 10 mg/kg to the levels of 126-130% of the control. Microsomal NADPH-cytochrome c reductase activities were decreased up to 67% of the control with the increasing dose of FK506 and to 62% in a group treated orally with cyclosporin A (25 mg/kg/d for 7 days), although another microsomal electron-transport component, cytochrome b5, was rather increased in all the treated groups. Treatment with FK506 or cyclosporin A did not reduce but slightly increased microsomal activities of aniline hydroxylation, p-nitroanisole O-demethylation and O-ethoxyresorufin O-deethylation. Microsomal depropylation of 7-propoxycoumarin, a typical P-450IIIA-substrate, was also not reduced in all dose groups of FK506, while it was decreased by the treatment with 25 mg/kg cyclosporin A.(ABSTRACT TRUNCATED AT 250 WORDS)


[The rebirth of the H1-antagonists].

[The rebirth of the H1-antagonists]. Anti H1s of the third generation, of which the first was Terfenadine, possess not only blocking effects on H1 receptors to histamine, but also have a "cromone-like" action on the membranes of cells that have been, or not, activated by specific stimuli (antigens). They act on the early phase of immediate hypersensitivity. These anti H1s of the new generation also have wider indications: asthma, perennial or pollen rhinitis, dermatoses such as urticaria and atopic dermatitis. Their tolerance has been remarkably improved with regard to the central effect (very reduced), such as absence of an atropine effect.


Transcripts encoding protein kinase C-alpha, -delta, -epsilon, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells. The protein kinase C (PKC) family of phospholipid-dependent serine-threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca(2+)-mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca2+. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta, but not PKC-beta or -gamma. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca2+. Interestingly, the PKC-zeta transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme. An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca2+ or the PKC activator 12-O-tetradecanoylphorbol-13-acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP-1, which contain an activating mutation of the c-Ha-ras gene. Like normal keratinocytes. 308 and SP-1 cells expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta. However, the abundance of PKC-zeta transcripts in both cell lines was reduced by 74-89% when compared with normal keratinocytes at similar Ca2+ levels. In addition, SP-1 but not 308 cells exhibited a sevenfold increase in PKC-eta mRNA when cultured in medium with 1.4 mM Ca2+. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v-Ha-ras oncogene. As with normal, 308, and SP-1 cells, v-Ha-ras keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta and -eta. The level of PKC-zeta transcripts was similar in normal and v-Ha-ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP-1 cells was not a direct result of ras activation. As in SP-1 cells, PKC-eta in v-Ha-ras keratinocytes was responsive to extracellular Ca2+, with a four-fold increase in transcript abundance in 0.12 mM Ca2+ medium relative to 0.05 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 400 WORDS)


Alterations in tumor angiogenesis associated with stable expression of the HIV tat gene. Recent evidence suggests that the human immunodeficiency virus type 1 (HIV) trans-activator gene (tat) has transforming properties and may be a causative factor in the development of certain types of cancers, in particular Kaposi's sarcoma (i.e., Vogel J. et al. Nature 335:606-611, 1988). To help elucidate the potential role or roles of the HIV tat gene in neoplastic transformation, cell lines were constructed that constitutively express a functional tat gene product. HeLa cells were coelectroporated with two plasmids, one containing the HIV tat gene in an expression cassette and another containing the dominant selectable marker gene xanthine guanine phosphoribosyltransferase (XGPRT). After XGPRT selection, single-cell clones that expressed a functional tat protein were identified by measuring chloramphenicol acetyltransferase (CAT) activity after electroporating a plasmid containing the CAT gene transcriptionally controlled by HIV trans-activation-responsive region (tar). Phenotypic alterations resulting from the expression of tat were then determined. Control cells and tat-expressing cells grew at similar rates in culture. However, when grown as tumors in nude mice, tat-expressing cells produced a lower percentage of tumors, and the tumors that were produced either regressed, stopped growing, or grew at a very reduced rate compared with cells not expressing tat. These differences may have resulted from a tat-associated reduction in neovascularization in the tumors. A comparison of total cellular proteins by two-dimensional polyacrylamide gel electrophoresis indicated only one reproducible alteration in a polypeptide of approximately 44 kDa and pl of approximately 6.2 associated with tat expression. These cells may be very useful in future in vitro and in vivo studies designed to examine the effects of HIV tat on endothelial and vascular smooth-muscle cells and the role of tat in the etiology of Kaposi's sarcoma.


Homologous and heterologous gap-junctional intercellular communication in v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial cell lines. We examined gap-junctional intercellular communication (GJIC) in a series of normal and v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial (RLE) cell lines using the scrape loading-dye transfer and fluorescence-recovery-after-photobleaching (FRAP) assays. Whereas the normal RLE cell line, the control helper virus-transduced cell line, and the v-myc-transduced cell line all showed excellent GJIC, the v-raf-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-raf/v-myc-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. Heterologous GJIC of these oncogene-transduced cell lines was also compared with that in the normal RLE cells. A modified FRAP assay, using fluorescent-microbead labelling to identify the oncogene-transduced cell from surrounding normal cells, was used to quantify the heterologous GJIC. The v-raf/v-myc-transformed RLE cells had no heterologous communication with the normal RLE cells, whereas v-raf- and v-myc-transduced cell lines maintained heterologous GJIC. Northern analysis showed that connexin 43 was the only gap-junction protein message expressed in these cell lines; connexin 32 and connexin 26 were not expressed. The levels of connexin 43 mRNA expression were relatively unchanged in all cell lines, suggesting that the reduction in GJIC was primarily at the posttranslational level. These findings suggest that reduction of homologous GJIC in v-raf- and v-raf/v-myc-transformed RLE cells is linked to their tumorigenic potential. Furthermore, the loss of heterologous GJIC, which we observed only in the v-raf/v-myc-transformed cells, might release such cells from the growth-regulating effects of surrounding normal cells, possibly contributing to their enhanced tumorigenic potential.


Differential induction of 12-O-tetradecanoylphorbol-13-acetate sequence gene expression in murine melanocytes and melanoma cells. We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281-3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of TIS gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.


Myelodysplastic syndromes. Pathogenesis, diagnosis and treatment. Our understanding of the biology of leukemia and myelodysplasia is still only partial. The diagnosis of myelodysplasia is often based on quantitative and qualitative findings in the peripheral blood and bone marrow. These findings are often shared by other disorders. There is a need for sensitive and inexpensive laboratory tests to determine clonality and karyotypic abnormalities in this disorder. Future classifications of these syndromes will need to be based on morphologic and biologic markers that are closely linked to disease progression, response to treatment, and survival. Our limited understanding of the pathogenesis of MDS decreases the specificity and effectiveness of our therapeutic interventions. Agents that are minimally toxic such as CRA, danazol, 1,25-dihydroxyvitamin D3, androgens, and pyridoxine are seldom useful. Antileukemic therapy and allogeneic bone marrow transplantation have a major role to play in patients younger than 45 years of age; in older patients these treatment modalities remain controversial because of their toxicity. Hematopoietic growth factors, used alone or in combination, may improve the quality of life and improve survival of patients with MDS. Growth factors may also decrease treatment-related mortality associated with chemotherapy and bone marrow transplantation and render these treatment modalities available for a higher percentage of patients. The development of more specific differentiating agents may permit hematopoietic differentiation while minimizing side effects.


Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel. Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.


Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration. The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.


Adhesion molecules on human lung dendritic cells and their role for T-cell activation. Human lung parenchyma contains potent accessory cells to stimulate T cells with many features of dendritic cells (DC), including a strong tendency to form aggregates with T cells. As contact phenomena may be crucial for T-cell activation, phenotypic and functional studies were conducted on adhesion molecules. DC from minced lung were isolated by their loose adherence and by their absence of autofluorescent inclusions by flow cytometry. DC compared with peripheral blood monocytes (Mo) show an increased density of Class I and II major histocompatibility antigens. Unexpectedly, the leukocyte integrins (beta 2 group) are decreased in density on DC. CD11a (LFA-1) and CD11b are less expressed, whereas CD11c is preserved. In contrast, the beta 1 chains are denser on DC with an increase in the alpha 5 chain (CD49e) on their surface. The alpha 4 chain of this later group (CD49d) is only weakly present and its ligand VCAM (INCAM-110) is not detected. DC-induced allogeneic T-cell proliferation is suppressed by antibodies against both the beta 1 and the beta 2 chains with a slight additive effect. Among the adhesins, LFA-3 and ICAM-1 are increased on DC compared with Mo. Antibodies against LFA-3, and to a lesser extent against ICAM-1, block T-cell activation. These data improve our knowledge of the phenotype expressed by DC and provide some clues as to how DC adhere with T cells and may transduce signals for T-cell activation by cell-cell interactions.


Immunohistochemical localization of epidermal growth factor and acidic and basic fibroblast growth factors in postnatal developing and adult rat lungs. Histologic preparations of lungs form 1-, 5-, 10-, 18-, and 25-day-old rats and adult rats were probed immunohistochemically with specific antibodies for the distribution of epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF). Immunoperoxidase staining of sections of adult rat lungs with a rabbit polyclonal antibody to bovine EGF was strong in ciliated cells of airways and mast cells, nonciliated cells of bronchioles, smooth muscle, type II cells of alveoli, and interstitial and epithelial cells of alveolar septal regions. Developing postnatal lungs had moderate and somewhat diffuse immunoreactivity in all epithelial cells, and more intense staining in vascular smooth muscle. Immunoperoxidase staining of adult rat lungs with a rabbit polyclonal antibody against bovine aFGF was distributed in identical fashion to EGF, with the exception of mast cells which were not reactive. These same sights were localized using an immunoperoxidase sequence with a rabbit polyclonal antibody to the leu 60-leu 98 fragment of aFGF (aFGFfr) with less background. In postnatal developing lungs, immunoreactivity with aFGF and aFGFfr preparations was diffuse and moderately intense in epithelial cells and vascular smooth muscle. Immunoperoxidase staining of adult rat lung sections with a monoclonal antibody to bovine bFGF was strong in hyaluronidase-digested preparations. Reactivity was principally confined to alveolar and vascular basement membrane regions and external laminae of smooth muscle. In early postnatal development, immunoreactivity for bFGF was found in basement membranes beneath developing epithelium and the endothelium of vessel wall intima and in the adventitia, with reactivity increasing with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)


Control of nucleocytoplasmic transport.

Control of nucleocytoplasmic transport. The movement of macromolecules between the nucleus and cytoplasm is tightly controlled. In the past few years it has become increasingly apparent that nuclear traffic is regulated not only by recognition of specific signals on proteins and RNAs, but also by cellular factors that modulate the efficacy with which these signals are recognized.


An immunohistochemical study on neurons and paraneurons of the pre- and post-natal chicken lung. The indirect immunoperoxidase (PAP) method was used on chicken lung specimens from embryos ranging in age from 6 days to hatching, chicks and adult chickens of up to 6 months. The ontogenesis and distribution of neurons and paraneurons containing immunoreactivities for serotonin (5HT), bombesin, vasoactive intestinal polypeptide (VIP), substance P (SP) and galanin were investigated. Serotonin-immunoreactive paraneurons were first detected in the pulmonary mesenchyma of 8-day-old embryos, while in the 12-day-old embryos the following neurons and paraneurons were first detected in their respective locations: serotonin-immunoreactive paraneurons in the bronchial epithelium; VIP- and galanin-immunoreactive ganglionic cells and SP-immunoreactive nerve fibres in the intrapulmonary ganglia. At hatching, serotonin-immunoreactive paraneurons in the epithelium of the air capillaries and air sacs, and bombesin-immunoreactive paraneurons in the epithelium of the primary bronchus, VIP-, galanin- and SP-immunoreactive nerve fibres in the lamina propria of the primary and secondary bronchi and in the pulmonary septa could also be shown. Some serotonin-immunoreactive small paraneurons were also found in the intrapulmonary ganglia. In the adult specimens, VIP-, galanin- and SP-immunoreactive nerve fibre networks were observed throughout the primary bronchus wall and in the lung septa. In intrapulmonary ganglia, VIP- and galanin-immunoreactive neurons and serotonin-immunoreactive small paraneurons could be more numerously demonstrated. Moreover, bombesin paraneurons occurred in the epithelium of primary and secondary bronchi, and serotonin-immunoreactive paraneurons were found in the epithelia of the bronchi and air sacs and in some pluricellular bodies in the lamina propria of the air sac ostia.


Expression of the interleukin-2 receptor on human fibroblasts and its biological significance. In this study, we have investigated the expression of the alpha and beta chains of the IL-2 receptor (IL-2R alpha, IL-2R beta) both at the membrane and at transcriptional levels during the lifespan of human embryonic fibroblasts. Here we show that the mAbs IOT14 and MIK beta 1 directed against the IL-2 binding sites of the IL-2R alpha and IL-2R beta respectively, stain human embryonic fibroblasts early in their life span. Data from [125I]rIL2 cross-linking experiments show the simultaneous expression of two IL-2 binding peptides of 70 and 55 kDa respectively on embryonic young fibroblasts as on lymphoid activated cells. The p55 and the p70 IL-2 binding peptides are shown to be specific for the IL-2R alpha and to the IL-2R beta by the finding that these bands are abolished by excess amounts of cold IL-2 and mAbs directed against the IL-2 binding sites of the alpha and beta chains. Scatchard analysis after [125I]IL-2 labelling shows the presence of both high affinity (150 sites with a Kd of 147 pM) and low affinity (1100 sites with a Kd of 4 nM) IL-2 binding sites. Northern blot and dot blot analysis show the presence of specific transcripts for the IL-2R alpha and IL-2R beta genes in early passaged fibroblasts. By contrast, in senescent cultures, only the IL-2R beta transcript were detected. Finally, IL-2 at low concentrations (36 pM) down modulates the level of the intercellular adhesion molecule ICAM-1 in young but not in senescent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)


High affinity for class II molecules as a necessary but not sufficient characteristic of encephalitogenic determinants. A direct binding assay specific for IAs molecules has been developed and its immunological relevance validated by examining, for a panel of nine different synthetic peptides, the correlation between their capacity to bind purified IAs and to inhibit IAs-restricted antigen presentation. The IAs assay thus developed has then been used to study the IAs binding affinity of a set of overlapping peptides spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IAs molecules have also been found to be high IAs binders, suggesting that variables other than MHC affinity are also involved in determining the pathogenic potential of self-derived determinants.


Renal cell carcinoma.

Renal cell carcinoma. The behavior of renal cell carcinoma remains one of the most unpredictable of the genitourinary neoplasms. Once this disease has spread beyond the confines of the kidney, it is extremely difficult to control. This year, emphasis has focused on the characteristic cytogenetic and chromosomal changes that are seen in this tumor that help to explain partially its enigmatic behavior. Immunotherapy remains the mainstay of nonsurgical therapy. Recent studies have examined the efficacy of using combinations of interferons, interleukin-2, or specific subpopulations of lymphoid cells to control metastatic renal cell carcinoma. The role of surgery in metastatic disease, tumor extending into the vena cava, and parenchyma-sparing operations continues to be examined. This review examines the most recent literature on each of these aspects in the treatment of this difficult and challenging tumor.


Prostatic cancer: are we closer to rational treatment selection? Despite the controversies in management for all stages of prostatic cancer, guidelines are emerging that allow for better selection of treatments for individual patients. For early stage disease, prostate-specific antigen determinations in conjunction with other staging procedures have refined our ability to define truly organ-confined disease. The more widespread use of laparoscopic lymph node dissections has spared many patients needless laparotomies. For patients with metastatic disease, the overall effect of potency-sparing antiandrogens as monotherapy needs to be investigated. Most encouraging is that more groups are using prostate-specific antigen changes to assess disease activity and the rapid translation of recent laboratory investigations into the clinic. As our ability to predict the biologic potential of an individual patient's tumor is improved, more individualized treatment recommendations will be possible.


Metastatic tumors of the axial spine.

Metastatic tumors of the axial spine. Spinal metastases are the most common tumors of the axial spine. Symptomatic spinal metastases in children and adults differ in a number of respects including culpable primary tumors, the degree of spinal column involvement, surgical strategies, and results of treatment. Magnetic resonance imaging has become the imaging method of choice for spinal tumors. The management of symptomatic spinal metastases is undertaken to relieve pain and to preserve or restore neurologic function. Radiation therapy is generally regarded as the initial treatment of choice. However, there has been a steady evolution in concepts and execution of surgical strategies for spinal secondaries. A prospective, randomized study should be undertaken to clarify the respective roles of therapeutic irradiation and surgery for metastatic tumors of the axial spine.


Heart preservation: analysis of cardioprotective infusate characteristics. Membrane stabilization, calcium antagonism, and protease inhibition on myocardial viability: a biochemical, ultrastructural, functional study. Thirty-three canine hearts were isolated after initial cardioplegia and preserved for 6 hours in 4 degrees C saline solution with intermittent infusion of cardioprotective solution every hour. Reperfusion was observed for 2 hours under normothermic cross-circulation. Hearts were divided into five groups depending on the agent(s) added to the K(+)-Mg2+ cardioplegic solution (K(+)-Mg(2+)-CP) infused. Control hearts (n = 6) received K(+)-Mg(2+)-CP solution alone; group I (n = 7) received lidocaine, 200 mg/L, added to the K(+)-Mg(2+)-CP solution; group II (n = 7) received betamethasone (250 mg/L) added to the formula for group I; group III (n = 6) received diltiazem (200 micrograms/L) added to the formula for group II; group IV (n = 7) received aprotinin (150 KIU/L) added to the formula of group III. Coronary sinus MB fraction of creatine kinase level was significantly decreased at 60 and 120 minutes of reperfusion in group II, as was mitochondrial aspartate aminotransferase level at 2 hours of reperfusion. Lysosomal enzyme release decreased in group IV. Myocardial adenosine triphosphate levels and total adenine nucleotides showed no significant difference among the groups at the end of reperfusion; however, myocardial adenosine diphosphate and adenosine monophosphate levels during reperfusion increased significantly in group I, and myocardial adenosine diphosphate and adenosine monophosphate levels at the end of reperfusion in groups I and IV were significantly higher than those of the control. Calcium overload, which was lowest in group II, was not completely prevented during reperfusion in any group. Left ventricular end-systolic pressure volume relationship in group II showed the "best" functional recovery. In addition, the ultrastructure of the left ventricular myocardium was well preserved in all groups. These results suggest that membrane stabilization with lidocaine and betamethasone affords beneficial effects on myocardial biochemical and functional viability. Diltiazem appears to be less effective in preventing calcium overload during ischemia-reperfusion, and protease inhibition with aprotinin (150 KIU/ml) seems to be highly effective in suppressing lysosomal enzyme activation-release and maintaining myocardial adenosine diphosphate and adenosine monophosphate levels.


Current concepts in leptomeningeal metastasis. Leptomeningeal metastasis is a common neurologic disorder affecting the entire neuraxis in patients with metastatic systemic cancer and primary brain tumors. The clinical presentation of leptomeningeal metastasis is pleomorphic and commonly affects the cerebral hemispheres, cranial nerves, or spinal cord and its roots. Diagnosis is confirmed by the presence of malignant cells in the cerebrospinal fluid or by clinical and neuroradiographic pattern compatible with leptomeningeal metastasis. Treatment is palliative and is directed at the entire neuraxis combining involved-field radiotherapy and intra-cerebrospinal fluid drug administration. Regional chemotherapy pharmacokinetically is best administered by intraventricular instillation employing frequent drug administration, thereby insuring prolonged cytotoxic drug exposure. Chemotherapeutic drug distribution is dependent on bulk flow of cerebrospinal fluid. This review will summarize current information regarding the incidence, clinical presentation, laboratory findings, pathology, pathophysiology, staging, treatment, and survival of patients with leptomeningeal metastasis.


[Sepsis caused by Mycobacterium tuberculosis in patients with AIDS]. Techniques for detecting mycobacteria in blood can be a good alternative for diagnosing tuberculosis in acquired-immunodeficiency syndrome patients (AIDS). Eleven patients with AIDS and pulmonary tuberculosis, and four controls with AIDS and without tuberculosis were studied. The diagnosis of mycobacteremia was made by the identification of the microorganisms in blood. The method used was a lysis-centrifugation with subsequent stain or culture identification in different media (agar Löwenstein-Jensen, M7H9, M7H11, brain-heart infusion and Sauton broth). In one case, direct staining of sample was positive. The culture results were positive in all 11 patients with tuberculosis and in none of the controls. M7H11 was the media that allows the fastest detection. This technique can be a good alternative method for tuberculosis diagnosis in AIDS patients. Is easy to perform, relatively fast an with diagnostic performance. It also could be of interest in extra-pulmonary tuberculosis.


The impact of FK506 on graft coronary disease of rat cardiac allograft--a comparison with cyclosporine. We studied the impact of FK506, a potent immunosuppressant, on graft coronary disease and graft-infiltrating lymphocyte subset after rat heart transplantation. Fisher rat heart grafts transplanted into Lewis rat recipients were divided into three groups: control (n = 7), rats treated with FK506 at a dose of 0.32 mg/kg/day intramuscularly (n = 7), and rats treated with cyclosporine at a dose of 10 mg/kg/day intramuscularly (n = 7). Grafts were removed on day 71 in the treated groups and on rejection in the control group. We blindly scored graft rejection and graft coronary disease on a scale of 0 to 4. Graft-infiltrating lymphocytes were investigated by flow-cytometric analysis with the following monoclonal antibodies: W3/25, anti-helper T lymphocyte; OX8, antisuppressor and cytotoxic T lymphocyte; and OX39, antiinterleukin-2 receptor. No difference of graft rejection was found between the two treated groups (FK506 1.66 +/- 0.49 versus cyclosporine 1.45 +/- 0.37), but the FK506 group showed severe graft coronary disease (FK506 2.14 +/- 0.82 versus cyclosporine 0.78 +/- 0.17; p less than 0.01) in this model. In flow-cytometric analysis, we found an increased proportion of OX8-positive lymphocytes (FK506 25.7 +/- 6.4 versus cyclosporine 4.9 +/- 2.4, p less than 0.01). These results suggest that suppression of cytotoxic T lymphocytes may be involved in graft coronary disease.


Monoclonal antibodies to soluble human TNF receptor (TNF binding protein) enhance its ability to block TNF toxicity. A soluble extracellular fragment of the human 55-60 kDa tumor necrosis factor receptor (sTNF-R I), originally isolated from urine, binds both TNF-alpha and TNF-beta and blocks the activity of these cytokines in biological assays. Three monoclonal antibodies (mAbs) raised against sTNF-R I (TBP-1, -2 and -6) as well as a mAb developed by immunization with the intact receptor (H398) were analysed for their epitope specificities in ELISAs and for biological activity in cytotoxicity assays on murine L-M cells. TBP-2 and H398 bind to related epitopes on sTNF-R I; they compete with TNF-alpha for binding and block the protective effect of sTNF-R I in the bioassay. MAbs TBP-1 and TBP-6 recognize two further, independent epitopes; both bind sTNF-R I in the presence of an excess of TNF-alpha. Both TBP-1 and TBP-6 markedly enhance the ability of sTNF-R I to protect cells against the cytotoxic activities of TNF-alpha and TNF-beta, but have no activity in the absence of sTNF-R I. Fab fragments show much lower activity. We propose that the ability of certain mAbs to enhance the protective activity of sTNF-R is due to a steric hindrance phenomenon.


Abrogation of the antiproliferative activity of oncostatin M by a monoclonal antibody. Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.


Distribution kinetics of FK-506, a novel immunosuppressant, after intravenous administration to rats in comparison with cyclosporin A. The distribution kinetics of a novel potent immunosuppressant, FK-506 (FK) has been studied in comparison with cyclosporin A (CyA) both in vivo and in vitro using blood specimens. The infusion studies on FK, 5.0 mg kg-1 through the portal and femoral veins showed that the mean hepatic extraction ratio of FK was 27.9 per cent. The effect of clamping both the hepatic artery and the portal vein on the plasma disappearance profiles of FK, 5.0 mg kg-1, and CyA, 3.5 mg kg-1 was studied. The plasma disposition kinetics of CyA was almost the same as in the normal rats. However, the plasma FK levels were about 10 times higher than those obtained in the control group rats. This difference is attributed to the restricted initial distribution of FK to the liver, because the volume of the initial distribution space, V1, of FK was about 10 times smaller than that obtained in normal rats. In in vitro experiments, drug distribution was studied in blood samples (2.0 ml) spiked with FK or CyA, 1.0 micrograms ml-1. The plasma drug levels measured at 2 min after drug administration were 0.842 +/- 0.012 micrograms ml-1 and 0.769 +/- 0.047 micrograms ml-1 for FK and CyA, respectively. The distribution volume in the blood compartment, VB, was determined by dividing the spiked amount of drugs with these plasma concentrations. The VB was 2.38 +/- 0.04 ml for FK and 2.62 +/- 0.16 ml for CyA. There was no significant difference in VB between FK and CyA. The plasma free fraction, fp of the drugs was measured by the equilibrium dialysis method. For FK, the mean fp values (+/- SE) were 1.31 +/- 0.18 per cent (2.0 micrograms ml-1) and 1.93 +/- 0.18 per cent (5.0 micrograms ml-1). For CyA, the fp values were 4.85 +/- 0.36 per cent (1.0 micrograms ml-1) and 5.75 +/- 0.82 per cent (5.0 micrograms ml-1). The hydrophobicity parameter, logP' determined through the HPLC method was 0.386 for FK and 0.545 for CyA. Although FK was less hydrophobic than CyA, its protein binding was higher than CyA.


'In vitro' action of histamine and cimetidine on amylase secretion in the rat pancreas. We report here the effect of histamine on amylase secretion in isolated lobules from the rat pancreas. Different concentrations of histamine increased amylase secretion, while the stimulatory effect was reduced by cimetidine although not by chlorpheniramine. These findings suggest that pancreatic lobules have H2 receptors. On the other hand, bethanechol induced a stimulatory effect on pancreatic lobules that was inhibited by cimetidine. Vibrio cholerae toxin stimulated amylase secretion, but this effect was not reduced by cimetidine. Cimetidine may thus block the stimulated amylase secretion interfering with the Ca2+ messenger system.


Cyclosporin A, FK506, rapamycin: the use of a quantitative analytic tool to discriminate immunosuppressive drug interactions. Potent immunosuppressive agents display toxic complications at full therapeutic doses: cyclosporin A (CsA) produces pleiotropic mesenchymal effects with prominent vasculopathy, whereas FK506 displays severe neurotoxicity associated with a similar range of mesenchymal and possibly vasculitic reactions. The concept of drug synergy seeks to exploit combinations of agents that promote each other's immunosuppressive effects. This article presents a quantitative method to assess synergy in vitro and in vivo--the median-effect analysis. Application of this method revealed synergistic interactions of CsA with steroids, an additive effect with azathioprine, and antagonistic effects with FK506 and enisoprost. Clearly, a synergistic therapeutic combination can be clinically useful only if it is not associated with a similar potentiation of toxic complications. Clinical practice has documented the synergistic relation of CsA plus steroids and the antagonistic relation of CsA with FK506 or enisoprost. Clinical trials of rapamycin, which displays extremely potent synergistic effects with CsA both in vitro with human and in vivo with animal immune responses, may afford important new insights for clinical immunosuppression.


Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses. We have recently described a new antitumor treatment, electrochemotherapy (ECT), based on the large local potentiation of the effects of the chemotherapeutic agent bleomycin (BLM) by electric pulses (EP) delivered at the tumor site. We demonstrate here that the host's immune response participates in cure achievement. We obtain an increase of the rate of completely cured animals by injecting mice with interleukin-2 (IL-2).


Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity. The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.


Differences in tyrosine phosphorylated proteins between cells expressing P210bcr/abl and P190bcr/abl. We analysed differences between the populations of tyrosine phosphorylated proteins in two cell lines, K-562 and MR-87, which express two different bcr-abl fusion gene products, using both immunoprecipitation and Western blotting with an anti-phosphotyrosine antibody. K-562 cells preferentially expressed P210bcr/abl (P210), while MR-87 expressed P190bcr/abl (P190). Tyrosine phosphorylated proteins with a molecular mass of 150 kDa (p150) and 115 kDa (p110) were found in both K-562 and MR-87. A 36 kDa protein (p36) was tyrosine phosphorylated in vivo only in K-562 cells, while proteins with a molecular mass of 140 kDa (p140) and 62 kDa (p62) were found only in MR-87 cells. Moreover, several proteins in the detergent-insoluble cell fraction were differently tyrosine phosphorylated in vitro in K-562 and MR-87 lysates. These results suggest that P210 and P190 may have different substrates, and thus, different signal transduction pathways for cell proliferation, although the differential association of such cellular proteins with the two bcr/abl products remains to be clarified.


Treatment of acute nonlymphocytic leukemia by combination of recombinant human granulocyte colony-stimulating factor and cytotoxic agents: a report of six cases. Six patients with recurrent and/or refractory acute nonlymphocytic leukemia (ANLL) were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) and cytotoxic agents administered simultaneously. Neither of the two patients who received cytosine arabinoside (ara-C) in combination with G-CSF achieved complete remission. The other four patients, who received multi-drug combination therapy together with G-CSF, all achieved complete remission. No major side effects due to G-CSF were observed. These results demonstrated that the effects of G-CSF in enhancing the sensitivity of leukemic cells to cytotoxic agents and accelerating the recovery of leukocytes could lead to its possible use in the treatment of ANLL.


Recombinant acidic human fibroblast growth factor (aFGF) stimulates murine megakaryocyte colony formation in vitro. Recombinant acidic human fibroblast growth factor (aFGF) significantly stimulated the formation of megakaryocyte colonies and the size of MK colonies as well as individual MKs in vitro in mice. When aFGF was combined with recombinant mouse interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-6 (IL-6) at their optimal doses, a synergistic action was found between aFGF and IL-3. The activity of aFGF could be completely abrogated by a monoclonal antimouse IL-6 antibody which specifically neutralized the action of mouse IL-6 but not human IL-6. These data indicate that aFGF provides positive growth signals of megakaryocyte progenitor cells, which can give rise to a synergistic action in the presence of IL-3 and which can be abrogated by the antimouse IL-6 antibody.


Expression of two types of kit ligand mRNAs in human tumor cells. The primary structure of human kit ligand (KL) growth factor mRNA was analyzed by polymerase chain reaction (PCR) and nucleotide sequencing. Two bands from the total RNAs of a variety of human tumor cell lines were amplified by RT-PCR. Nucleotide sequences of these cloned cDNAs revealed that an 84-nucleotide stretch, corresponding to a spacer chain which contains the site for proteolytic release of the cytokine domain, was missing in the shorter clone, resulting in a transmembrane-bound form of KL. The relative intensity of the two bands in human bone marrow and tumor cells was then determined. In the bone marrow cells, THP1 and HuH7, the band for the membrane-bound type of KL was relatively more intense, whereas the reverse was the case in Daudi, K562 and HT1080 cells, suggesting that there are variations in the expression level of these two human KL mRNAs.


Phenotypic changes induced by interleukin-2 (IL-2) and IL-3 in an immature T-lymphocytic leukemia are associated with regulated expression of IL-2 receptor beta chain and of protein tyrosine kinases LCK and LYN. We have previously reported the establishment of an interleukin-3 (IL-3)-dependent and phenotypically myeloid cell line (TALL-103/3), obtained by culturing cells from an immature T-lymphoblastic leukemia in the presence of IL-3. These cells differentiated into a T-lymphoid cell line (TALL-103/2) upon removal of IL-3 and incubation in IL-2. Despite the different phenotype, the two cell lines remained karyotypically and genotypically identical. Here, we have analyzed the phenotypic changes and the signaling events induced by these two lymphokines in TALL-103/3 cells by switching them to temporary growth in IL-2 and returning them to IL-3. All four sublines obtained (the myeloid in IL-3 and the lymphoid in IL-2) expressed RNA for CD3, IL-2 receptor (R) alpha, and T-cell receptor (TCR)-gamma and -delta chains. However, cells cultured in IL-3 failed to express detectable levels of the IL-2R beta chain at both the protein and RNA levels, whereas cells exposed to IL-2 always expressed IL-2R beta. In parallel with the changes in IL-2R beta expression, the SRC-like protein tyrosine kinase (PTK) p56 LCK could not be detected in IL-3-dependent cells, but was abundant in the IL-2-dependent cells and underwent markedly increased autophosphorylation in response to IL-2. In contrast, p53/p56 LYN was highly expressed in IL-3-dependent cells, and greatly decreased when these cells were switched to growth in IL-2. LYN kinase autophosphorylation modestly increased in response to IL-3. None of the other kinases in the SRC family that were tested underwent increased autophosphorylation after lymphokine stimulation, indicating the specificity of IL-2 for LCK and of IL-3 for LYN. The TALL-103 cell lines provide a unique system to study the interaction between lymphokines and SRC-family PTKs in signal transduction pathways leading to hematopoietic cell differentiation.


OMA-AML-1: a leukemic myeloid cell line with CD34+ progenitor and CD15+ spontaneously differentiating cell compartments. OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15-. Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self-renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration-dependent proliferative responses to interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.


Crossreacting human lymphoma idiotypes.

Crossreacting human lymphoma idiotypes. We have examined an unselected series of 72 lymphomas of diverse histologies with a panel of mouse monoclonal antibodies specific for human B-lymphoma-derived Ig idiotypes (anti-ids) to determine the nature and extent of id/anti-id crossreactivity. The anti-id antibodies were prepared from Ig isolated from seven follicular center cell lymphomas by heterohybridoma technique. Thirty-six of 75 individual anti-ids obtained in this manner were further selected based on their reactivity with highly restricted or private idiotypic determinants. Twelve of the 72 (17%) lymphoma biopsies reacted with one or more of the 36 anti-ids that detect private determinants. A pool consisting of five individual antibodies would have detected 11 of the 12 crossreacting tumors. Staining of tumor cell populations was homogeneous in the positive cases, suggesting uniform idiotype expression. If there was a segregated staining pattern, it was generally related to the presence of CD3+ T cells in the section. These follicular center cell-derived anti-ids cross-reacted with follicular center cell tumors of all histologic grades with frequencies ranging from 13% to 50%. The structural basis for the crossreactivity of our lymphoma-derived private anti-ids is as yet not known. However, the reactivity of certain anti-ids with both kappa- and lambda-expressing tumors suggests a biased use of V gene segments in these crossreactive clones that is probably related to the VH gene. These data suggest the possibility that lymphoma may develop in a highly restricted pool of normal differentiated B cells or in B-cell subsets that express a limited repertoire of unmutated VH gene segments.


Multiple Rh messenger RNA isoforms are produced by alternative splicing. Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.


Evidence that stem cell factor is involved in the rebound thrombocytosis that follows 5-fluorouracil treatment. The mechanisms responsible for 5-fluorouracil (5FU)-induced rebound thrombocytosis are not completely understood. SI/SI(d) mice, which do not undergo rebound thrombocytosis in response to 5FU, provide a genetic approach to the study of this phenomenon. Recent reports by several groups that the SI locus encodes a protein known variably as stem cell factor (SCF), mast cell growth factor, or kit ligand, suggests the possibility that the lack of wild-type SCF in SI/SI(d) mice is responsible for their defective response to 5FU-induced thrombocytopenia. It is shown in this report that SCF-treated SI/SI(d) mice are as capable as their wild-type littermates in undergoing rebound thrombocytosis. W/Wv mice, mutated at the locus encoding the SCF receptor, also do not undergo rebound thrombocytosis, but are not responsive to SCF treatment. In normal mice, it is shown by RNA solution hybridization that SCF mRNA expression is increased during the 5FU-induced platelet nadir period. It is also shown by autoradiography that maturing megakaryocytes express SCF receptors, and that in vivo administration of SCF significantly raises the numbers of megakaryocytes, as well as circulating platelet counts. Taken together, these data indicate that SCF may be an important regulator of platelet production under both normal and physiologically disturbed situations.


A c-kit ligand, recombinant human stem cell factor, mediates reversible expansion of multiple CD34+ colony-forming cell types in blood and marrow of baboons. The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte-monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.


Effects of bone marrow stimulatory cytokines on human immunodeficiency virus replication and the antiviral activity of dideoxynucleosides in cultures of monocyte/macrophages. Cells of the monocyte lineage are important targets for the replication of human immunodeficiency virus (HIV). Our group and others have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates HIV replication in monocyte/macrophages, but that it also enhances the anti-HIV activity of 2',3'-dideoxy-3'-azidothymidine (AZT). In the present study, we have explored the effects of other bone marrow stimulatory cytokines on the replication of HIV and on the anti-HIV activity of certain dideoxynucleosides in human peripheral blood monocyte/macrophages (M/M). Like GM-CSF, macrophage CSF (M-CSF) enhanced HIV replication in M/M. In contrast, granulocyte CSF (G-CSF) and erythropoietin (Epo) had no such effects. The anti-HIV activity of zidovudine (AZT) was increased in M/M exposed to GM-CSF. In contrast, the anti-HIV activity of AZT was unchanged in M/M exposed to M-CSF, and the activities of 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddl) were unchanged or slightly diminished in M/M stimulated with GM-CSF or M-CSF. These differential activities of AZT and ddC were paralleled by differential effects of the cytokines on the anabolism of these drugs to their active 5'-triphosphate moieties. GM-CSF increased the levels of AZT-5'-triphosphate (at least in part through an increase in thymidine kinase activity) and overall induced an increase in the ratio of AZT-5'-triphosphate/thymidine-5'-triphosphate. In contrast, M-CSF-induced increases in AZT-5'-triphosphate were roughly matched by increases in thymidine-5'-triphosphate. Also, GM-CSF- or M-CSF-induced increases in the levels of ddC-5'-triphosphate were associated with parallel increases in the levels of deoxycytidine-5'-triphosphate (the physiologic nucleoside that competes at the level of reverse transcriptase), so that there was relatively little net change in the ddC-5'-triphosphate/deoxycytidine-5'-triphosphate ratio. Thus, bone marrow stimulatory cytokines may have a variety of effects on HIV replication and on the activity and metabolism of dideoxynucleosides in M/M.


Calcium pools mobilized by calcium or inositol 1,4,5-trisphosphate are differentially localized in rat heart and brain. Calcium-induced calcium release (CICR) pools have been demonstrated in brain and heart microsomes biochemically and autoradiographically by the sensitivity of 45Ca2+ accumulation to Mg2+, ATP, ruthenium red, caffeine, and tetracaine. The CICR pool colocalizes with [3H]ryanodine binding sites, supporting the notion that [3H]ryanodine labels CICR pools. Sites of CICR pools in the brain contrast with those of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools with reciprocal localizations between the two Ca2+ pools in several structures. Thus, in the hippocampus CA-1 is enriched in IP3-sensitive Ca2+ pools, whereas CICR pools are highest in CA-3 and the dentate gyrus. The corpus striatum and cerebellum are enriched in IP3 pools, whereas the medial septum and olfactory bulb have high CICR densities. In cardiac tissue, CICR is localized to atrial and ventricular muscle, whereas IP3 pools are concentrated in coronary vessels and cardiac conduction fibers. The reciprocal enrichment of IP3 and CICR Ca2+ pools implies differential regulation of Ca2+ hemostasis in these tissues.


The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. PMR1, a Ca(2+)-adenosine triphosphatase (ATPase) homologue in the yeast Saccharomyces cerevisiae localizes to a novel Golgi-like organelle. Consistent with a Golgi localization, the bulk of PMR1 comigrates with Golgi markers in subcellular fractionation experiments, and staining of PMR1 by indirect immunofluorescence reveals a punctate pattern resembling Golgi staining in yeast. However, PMR1 shows only partial colocalization with known Golgi markers, KEX2 and SEC7, in double-label immunofluorescence experiments. The effect of PMR1 on Golgi function is indicated by pleiotropic defects in various Golgi processes in pmr1 mutants, including impaired proteolytic processing of pro-alpha factor and incomplete outer chain glycosylation of invertase. Consistent with the proposed role of PMR1 as a Ca2+ pump, these defects are reversed by the addition of millimolar levels of extracellular Ca2+, suggesting that Ca2+ disposition is essential to normal Golgi function. Absence of PMR1 function partially suppresses the temperature-sensitive growth defects of several sec mutants, and overexpression of PMR1 restricts the growth of others. Some of these interactions are modulated by changes in external Ca2+ concentrations. These results imply a global role for Ca2+ in the proper function of components governing transit and processing through the secretory pathway.


Antitumor action of retinoids: inhibition of tumor cell line-induced angiogenesis and prevention of tumors in mice. Acitretin was shown to inhibit angiogenic response to the tumorigenic SKV cell line bearing HPV16 genome and to sarcoma L-1 cell line, both in vitro and in vivo systems. The lowered angiogenic response to tumor cells was independent of duration and timing of the application of acitretin to animals. Acitretin, but not etretinate, was also found to be effective in the prevention of sarcoma tumors in mice.


Carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size. Comparison of the haemagglutinins (HA) of the pathogenic avian influenza viruses A/FPV/Dutch/27 (H7N7) and A/FPV/Rostock/34 (H7N1) revealed 94.7% nucleotide and 93.8% amino acid sequence homologies. Six of the seven N-glycosidic oligosaccharides of the Rostock HA are at the same positions as the six carbohydrates of the Dutch strain. The additional oligosaccharide side chain of the Rostock strain, which is of the complex type, is attached to asparagine149 in antigenic epitope B. The accessibility of this antigenic epitope has been analysed by using rabbit antisera raised against synthetic peptides comprising amino acids 143-162. The carbohydrates of the HA of the Rostock strain have been modified (i) to truncated cores by expression in insect cells using a baculovirus vector, (ii) to oligomannosidic side chains by growth in the presence of the trimming inhibitor methyldeoxynojirimycin and (iii) to a single N-acetylglucosamine residue by removal of the oligomannosidic sugar with endo-beta-N-acetylglucosaminidase H. Neither the authentic nor the modified oligosaccharides allowed antibody binding, as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Reactivity was observed, however, after complete removal of the carbohydrate from HA of the Rostock strain by digestion with peptide-N-glycosidase F. HA of the Dutch strain was reactive without prior peptide-N-glycosidase F treatment. These results demonstrate that a single N-acetyl-glucosamine at asparagine149 is sufficient to prevent recognition of the peptide epitope.


[Comparative study of chuanxiong and dextran 40 in the treatment of acute cerebral infarction]. This paper reports the results of a double-blind trial in 220 patients with acute cerebral infarction evidenced by CT, who were randomly divided into ligusticum chuanxiong group (134 cases) and low molecular weight dextran group (86 cases). A weighted scoring system was adopted to evaluate the neurologic function and living capability. The results showed that the total therapeutic efficacy rate in chuanxiong group and in dextran 40 group were 86.6% and 62.8% respectively. The effect of chuanxiong on the treatment of acute cerebral infarction was superior to low molecular weight dextran and the difference between the two groups was statistically significant (P less than 0.01).


[Prevention of post-transfusion non-A, non-B hepatitis by anti-HCV screening]. Since May 1990 a specific enzyme immunoassay (EIA) for NANBH has been developed by recombinant DNA technology which detects antibodies to a virus called hepatitis C virus (HCV). The anti-HCV EIA was manufactured by Ortho-Diagnostic Systems with recombinant antigens from Chiron Corp. based on extraction from high infectious titre chimpanzee plasma RNA after transcription into cDNA. We tested the anti-HCV prevalence of blood donors and hemodialysis patients. The anti-HCV prevalence with the first generation test was 0.52% (Ortho), 0.87% (Abbott) in blood donors and 4.16% in hemodialysis patients. The second generation anti-HCV test (Abbott) with improved sensitivity and specificity comprises 0.25% repeated anti-HCV positive blood donors and 8.2% anti-HCV positive hemodialysis patients.


The pea ferredoxin I gene exhibits different light responses in pea and tobacco. We monitored Fed-1 (encoding ferredoxin I) mRNA levels in etiolated transgenic tobacco seedlings containing the intact pea Fed-1 gene to determine if the characteristic light responses of this gene in pea seedlings are also observed in transgenic tobacco. Fed-1 transcript levels in transgenic tobacco seedlings closely paralleled those of the native gene in pea buds when etiolated seedlings were transferred to white light. However, the response to red light was much smaller in tobacco than in pea and was not efficiently reversed by far-red light. The red light response of endogenous tobacco ferredoxin transcripts is closely comparable to that of the Fed-1 transgene, with a similar lack of photoreversibility. Thus, the pea Fed-1 transgene responds normally to tobacco gene-regulatory factors, but these factors are less influenced by phytochrome in tobacco cotyledons than in pea buds.


Natural killer cell surface markers on cells containing parallel tubular structures. Parallel tubular structures (PTS) and/or the associated electron-dense granules, thought to contain molecules responsible for target cell lysis, can be detected in the cytoplasm of lymphocytes with large granular lymphocyte (LGL) morphology. In the present study we compared PTS presence in freshly isolated peripheral blood lymphocytes and peripheral blood lymphocytes incubated overnight in the presence of human pooled serum, sera from patients with Hodgkin's disease and interferon-alpha. Under all conditions we found PTS in the majority of CD16+ cells (64.3-74.8%) but less than 41.8% in CD57+ cells. In the case of double-labeled lymphocytes, 41.0-61.7% CD16+/8+ but only 24.2-27.5% CD57+/8+ cells were PTS+. Thus, in all cases where lymphocytes expressed CD16 antigen there was a high percentage of PTS positivity. Although the PTS+ cells exhibited phenotypic heterogeneity there was, except for a proportion of CD57+ lymphocytes which exhibited less of the characteristic LGL features, generally LGL morphological homogeneity. CD16 lymphocytes are potentially more cytotoxic than CD57 and CD8 cells. Taking this into consideration, the presence of the PTS in the majority of CD16 cells suggests an important role for PTS in target cell lysis.


Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids. Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.


The ciliary body--the third organ found to synthesize indoleamines in humans. Indoleamines are associated with circadian rhythms in pineal gland and retina. Because the ciliary epithelium has an embryonic origin similar to that of pineal gland and retina, and intraocular pressure shows circadian variations, indoleamines were searched for in aqueous humor and ciliary body in humans. In aqueous humor, serotonin, 6-hydroxymelatonin, and melatonin were simultaneously detected and measured using high-performance liquid chromatography with electrochemical detection. The concentration was 48.7 +/- 10.9 ng/ml for serotonin, 0.47 +/- 0.8 ng/ml for melatonin, and 13.9 +/- 7.7 ng/ml for 6 hydroxymelatonin. In ciliary bodies from freshly enucleated human eyes, tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid were detected using high-performance liquid chromatography with simultaneous fluorescence- and electrochemical detection. Finally, the enzymatic activities of arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), enzymes indispensable in the synthesis of melatonin, were measured. The NAT activity was 273 +/- 25 pmol/mg protein/hour and that of HIOMT, 13520 +/- 50 pmol/mg protein/hour in ciliary body. Comparison of these activities (NAT versus HIOMT) permits the suggestion that NAT is a limiting enzyme in serotonin metabolism in this tissue. These findings indicate that a circadian rhythm of indoleamines exists in human aqueous humor and that the human ciliary body is the third organ, after the pineal gland and the retina, found to synthesize indoleamines in humans.


Expression of GABAA receptor polypeptides in clonal rat cell lines. The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [3H-]muscimol binding; none bound [35S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed alpha subunits. Northern blot and polymerase chain reaction analysis showed that out of alpha 1, alpha 4, beta 1 and gamma 2 subunits, only alpha 1 subunit mRNA was present. Thus, GABAA-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.


Intrachoroidal dye leakage in indocyanine green fundus angiography after experimental commotio retinae. Dye leakage in sodium fluorescein (FLUO) fundus angiography indicates damage to the blood-retinal barrier. However, dye leakage in indocyanine green (ICG) fundus angiography does not mean the same, because of the larger molecular size of the dye and impermeability of the choroidal vessels to it. The possibility of dye leakage in ICG angiography has not yet been revealed in an experimental study in which the blood-retinal barrier is undamaged. We report here that intrachoroidal dye leakage may occur in ICG angiography in an experimental model of the traumatic retinal opacity of the rabbit eye, even when the blood-retinal barrier is undamaged. This mechanism of dye leakage in ICG angiography is quite different from the leakage of FLUO angiography. Pathological choroidal vessels with increased permeability, such as choroidal neovascularization under the retinal pigment epithelium, can be observed using ICG angiography.


Failure of a protein synthesis inhibitor to modify glutamate receptor-mediated neurotoxicity in vivo. The delayed neuronal death (DND) resulting from brief forebrain ischemia has recently been reported to be markedly attenuated by parenteral administration of the reversible protein synthesis inhibitor, anisomycin. Previous work suggests that ischemia-induced DND is mediated by glutamate acting at one or more glutamate receptors, since glutamate receptor antagonists have been reported to reduce ischemia-induced DND. Consequently, we tested whether anisomycin could modify DND induced by direct intracerebral administration of the excitotoxins, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxasole (AMPA) or kainic acid. Anisomycin, administered parenterally, in multiple doses did not alter DND induced by any of these excitotoxins, nor did combined parenteral and direct intracerebral injection of anisomycin protect against DND induced by AMPA. Thus, neurotoxicity induced by direct intracerebral administration of NMDA, AMPA or kainic acid does not appear to require de novo protein synthesis, and, therefore, is not likely to be mediated by the expression of a programmed cell death cascade.


Patterns of glucose use after bicuculline-induced convulsions in relationship to gamma-aminobutyric acid and mu-opioid receptors in the ventral pallidum--functional markers for the ventral pallidum. Bicuculline-induced convulsions increased glucose use throughout the brain and sharply demarcated the ventral pallidum and globus pallidus. Glucose use in the nucleus accumbens also increased after bicuculline-induced convulsions, except for a circumscribed region in the dorsomedial shell. Since the projection from the nucleus accumbens to the ventral pallidum contains gamma-aminobutyric acid (GABA) and the opioid peptide, enkephalin, the pattern of increased glucose use in the ventral pallidum and nucleus accumbens after bicuculline-induced convulsions was compared to the topography of GABAA and mu-opioid receptors. The pattern of glucose use in the nucleus accumbens and ventral pallidum resembled the topography of GABAA, but differed from that of mu-opioid receptors. Bicuculline may disinhibit GABAergic efferents to the ventral pallidum resulting in a dramatic increase in glucose use within striatopallidal synaptic terminals as well as in local terminals of the pallidal projection neurons.


Imipramine inhibits intrathecal substance P-induced behavior and blocks spinal cord substance P receptors in mice. The mechanism of the antinociceptive effect of the tricyclic antidepressant imipramine was investigated in mice. Intrathecal (i.t.) administration of imipramine produced dose-dependent antinociception in the tail-pinch and tail-flick tests with ED50 values (95% confidence limit) of 27.5 (17.0-43.9) and 20.2 (12.6-32.2) nmol, respectively. In substance P (SP)-induced nociceptive behavior, imipramine (i.t.) also produced dose-dependent antinociception with ED50 value of 20.2 (16.1-25.2) nmol. Tissue concentration of imipramine was between 55.2 and 104.4 nmol/g tissue when these ED50 values of imipramine were i.t. administered. In the SP-induced behavior, the antinociceptive effect of 31.6 nmol of imipramine was not antagonized by the alpha-adrenergic receptor antagonist phentolamine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In vitro study, imipramine dose-dependently inhibited specific [3H]SP binding in the spinal cord homogenate with IC50 value of 2.37 x 10(-4) M and this value corresponds to 8.6 mumol/g tissue concentration. These data indicate that imipramine produces antinociceptive effect at about 100 times lower dose than SP receptor blockade.


Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation. In vitro superfusion was used to examine the effect of inflammation on the release of immunoreactive calcitonin gene-related peptide (iCGRP) and substance P(iSP) from the dorsal horn of rats. Three hours after carrageenan, hindpaws exhibited hyperalgesia, edema, and hyperthermia. Spontaneous and capsaicin-evoked release of iCGRP and iSP were significantly increased following inflammation. The enhanced release of iCGRP and iSP in the dorsal horn may serve as a biochemical marker for the development of hyperalgesia.


Serotonin denervation enhances responsiveness of presynaptic dopamine efflux to acute clozapine in nucleus accumbens but not in caudate-putamen. Clozapine alters mesolimbic dopamine (DA) function but spares nigrostriatal DA function in laboratory animals, but the underlying mechanism is unknown. In the present study, acute intraperitoneal injection of clozapine (5-40 mg/kg) increased extracellular DA levels in nucleus accumbens (Acb) and caudate-putamen (CPu) of awake, freely moving rats as measured by in vivo brain microdialysis, without anatomic selectivity. However, in serotonin (5HT)-denervated rats acute clozapine preferentially enhanced DA levels in Acb as compared to CPu. Since (i) up-regulation of 5HT receptors on DA neurons may result from 5HT denervation, (ii) clozapine has potent anti-5HT action, and (iii) 5HT receptors are more dense in Acb than CPu, these data appear to add additional weight to previous suggestions that a serotonergic mechanism may partly underlie clozapine's mesolimbic selectivity.


Co-localized but target-unrelated expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons after peripheral nerve crush injury. Expression of vasoactive intestinal polypeptide (VIP) and galanin in dorsal root ganglion (DRG) neurons is known to be induced by peripheral nerve injury. We investigated (1) whether VIP and galanin were co-expressed by DRG neurons and (2) whether such neurons innervated specified peripheral targets (visceral, cutaneous or muscular). An antibody to the 200 kDa neurofilament subunit (NF200) was used as a marker for large type-A cells in the DRG. VIP and galanin were respectively observed in 22% and 67% of DRG neurons at the L5 spinal level after crushing of the sciatic nerve. Most VIP-containing neurons were small type-B cells (about 90%) and approximately 95% of VIP-containing neurons also showed galanin-like immunoreactivity. Galanin was expressed by both large type-A and small type-B cells. Immunocytochemistry combined with a retrograde tracer revealed that about 70-80% of the small type-B cells in each sensory division displayed VIP-like immunoreactivity, and that most of the tracer-labeled neurons also expressed galanin. These findings suggest that the expression VIP and/or galanin in response to peripheral nerve crush injury is a property common to visceral, cutaneous and muscular sensory neurons.


Mesencephalic type I astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture. We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)


Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats. Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Galanin in the medial septal area impairs working memory. Galanin, a peptide of 29 amino acids, is co-localized with acetylcholine in a subpopulation of neurons of the medial septal area (MSA) that project to the hippocampus. Galanin reverses the actions of acetylcholine in several biochemical and behavioral procedures, and may be involved in memory processes. To test the possibility that galanin acts on the cell bodies of MSA neurons, two measures of septohippocampal function were assessed following intra-septal microinfusion of galanin or two synthetic fragments of galanin (1-16 and 21-29). The behavioral measure was choice accuracy in a working memory task in a T-maze. The electrophysiological measure was hippocampal theta activity recorded from the dentate hilus. The galanin fragment, 1-16, and the complete peptide, 1-29, decreased choice accuracy and decreased hippocampal theta activity in a dose-dependent fashion. Saline and the 21-29 fragment had no effect on choice accuracy and hippocampal theta. Sensorimotor performance was unaffected. These findings demonstrate that galanin impairs working memory when administered directly into the MSA and suggest that galanin inhibits MSA neural activity.


Risperidone: clinical development: north American results. This multicenter trial indicates that risperidone is an effective treatment for the positive and negative symptoms of schizophrenia. The most effective dose appears to be about 6 mg of risperidone daily. This dose was more effective than 20 mg of haloperidol. In addition, this dose was associated with very low rates of extrapyramidal side effects.


Influence of the culture medium on the synthesis of alpha-D-glucans by Streptococcus cricetus AHT. Three different alpha-D-glucosyltransferases (GTFs) were separated from culture filtrates of Streptococcus cricetus strain AHT grown in a complex, standard medium in batch culture or under defined conditions of growth in the chemostat. Two of the enzymes (GTF-S1 and GTF-S2) converted sucrose into branched, soluble dextrans, and the third (GTF-I) produced a relatively linear, water-insoluble, predominantly (1----3)-linked alpha-D-glucan. When the organism was grown in complex medium modified by the removal of the fraction of high molecular weight, only GTF-S1 and GTF-S2 were released, and no GTF-I was detected. The water-insoluble glucan fraction obtained by incubating the cell-free filtrate with sucrose contained from 17 to 25% of (1----3)-glucosidic linkages, and accounted for up to 78 and 4% of the total glucans derived from growth in standard and modified medium, respectively. The soluble glucans produced in the same reaction were fractionated with ethanol to give, from both media, two distinct dextrans comprising (1) a highly branched dextran similar to the S1-dextran product of GTF-S1 and (2) a dextran containing fewer branch linkages and up to 86% of (1----6)-alpha-D-glucosidic linkages. A GTF responsible for the synthesis of the latter dextran was not separated. The structures of the glucan fractions and the products of the separated GTF were examined by enzymic degradation and methylation analysis.


Action patterns of amylolytic enzymes as determined by the [1-14C]malto-oligosaccharide mapping method. A valuable technique for oligosaccharide mapping, utilizing radioactive malto-oligosaccharides, multiple-ascent p.c., and radioautography, has been developed for identifying the action patterns of the glucoamylase isozymes, alpha-amylases, beta-amylase, glucosyltransferase, and glucanosyltransferase. The glucoamylase isozymes act by multi-chain mechanisms on malto-oligosaccharides and most likely on starch and glycogen. The alpha-amylases act endo-wise and randomly hydrolyze alpha-(1----4)- but not alpha-(1----6)-glucosidic bonds. These amylases may act by single-chain and/or multi-chain mechanisms, depending on the number of hydrolytic attacks per single encounter of the enzyme and the substrate. The beta-amylases hydrolyze malto-oligosaccharides by a multi-chain mechanism. A fungal glucosyltransferase from Aspergillus niger transfers glucose units by a single-chain mechanism from maltose to glucosyl acceptors to yield new gluco-oligosaccharides with alpha-(1----4) and alpha-(1----6) linkages. A novel type of transferase isolated from Bacillus subtilis acts by a multi-chain mechanism and transfers segments of 2 to 5 glucose residues from malto-oligosaccharides to acceptor co-substrates. An alpha-amylase from the same organism removes maltotriose units from the non-reducing ends of oligosaccharides by a multi-chain mechanism.


A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro. EM radioautographic study on RNA synthesis in aging mouse spleen was conducted after 3H-uridine labeling in vitro. The localization of radiolabelled precursor was used to determine the site of RNA synthesis. The site of the radiolabelled uridine uptake was localized in the haematopoietic cells, particularly in the lymphoblasts. In the labelled cells, most of the silver grains were localized in the nucleus, specifically in the euchromatin. Few cytoplasmic organelles such as the mitochondria and endoplasmic reticulum were labelled with 3H-uridine. Silver grains were also observed over the nucleoli. The labeling index was expressed as the percentage of labelled cells over the total number of cells counted. The labeling index increased from day one after birth and progressively until the 14th day. Thereafter, the labeling index decreased gradually until the 10th month. A significant difference of p less than 0.05 was noted. In all the EMRAG analyzed, it was observed that the number of silver grains per cell increased proportionally with the labeling index. The result of the quantitation of the changes in RNA synthesis correlated well with the maturational development/aging of the animal.


Inter-alpha-trypsin inhibitor-related immunoreactivity in human tissues and body fluids. The distribution of inter-alpha-trypsin inhibitor (ITI) and related inhibitors was investigated in normal human tissues and body fluids by using an enzyme-linked immunosorbent assay (ELISA) and a streptavidin-biotin-peroxidase immunohistochemical technique. ITI-related immunoreactivity was localized in different cell types of various organs, such as liver, kidney, testis, gross intestine, cutis and brain. Specific immunoreactivity was also detected in serum, urine and bronchial mucus. This widespread, but not ubiquitous pattern of localization suggests that, in addition to the well known plasmatic role, ITI and/or ITI-related inhibitors may play a number of different physiological roles in various human tissues.


Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats. Normal epithelial cell differentiation is characterized by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokeratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5 + 8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5 + 8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinized squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of vitronectin in embryonic rat endocardial cell migration in vitro. Vitronectin is one of the extracellular matrices that mediate cell spreading and attachment in vitro. In the present paper, we demonstrate the involvement of vitronectin in the migration of cushion mesenchymal cells of the embryonic rat heart. Immunohistochemistry established the localization of vitronectin in the myocardial cells and in some of the cushion mesenchymal cells of the truncus arteriosus and atrioventricular canal. In vitro, vitronectin, fibronectin, and collagen type-I revealed significant stimulating activity for cushion mesenchymal cell migration. The distance migrated by cushion mesenchymal cells cultured on vitronectin, collagen type-I, or both vitronectin and fibronectin was similar, but that on fibronectin was significantly shorter. Following the addition of anti-vitronectin IgG to the medium, the migration distance of cushion mesenchymal cells on fibronectin was remarkably increased. Most explants on vitronectin or on both vitronectin and fibronectin became detached from dishes after the addition of the antivitronectin antibody. Immunostaining revealed that cushion mesenchymal cells cultured on substrata other than vitronectin synthesized vitronectin. From these results, it is suggested that vitronectin is synthesized by myocardial cells and some cushion mesenchymal cells, and that vitronectin inhibits cell movement on fibronectin. This feature of vitronectin may be important in the regulation of the migration of cushion mesenchymal cell in vivo.


[An immunohistochemical and ultrastructural study of 20 chordomas]. Altogether 20 chordomas were reported. The site distribution included 9 cases at the sacrococcygeal region, another 9 cases at the spheno-occipital region, 1 at the cervical vertebra, and another 1 at the lumbar vertebra. Histologic examination revealed that characteristic "physaliphorous cells" were easily identified in all the 19 cases. Tissue for immunohistochemistry study was available in 18 cases. Among them, tumor cells were found strongly positive to EMA, but negative for CEA. 16/18 cases also showed positive for keratin and S-100 protein. Totally, 2 cases were studied ultrastructurally and there were abundant RER and microfilaments seen in the cytoplasm of the tumor cells but only few surface microvilli detected. The epithelial nature of chordoma is strongly supported by the ultrastructural and immunohistochemical findings of these 20 cases.


[Application of microwaves in special and immunohistochemical staining]. Special and immunohistochemical staining are commonly used in clinicopathological diagnosis. However, the processing of most staining methods takes quite a long time even to 1-2 days. Application of microwaves to the staining process such as for nerve myelin, striated muscle fibers, acid mucin, melanin, hemosederin, aspergillus, basement membrane, HHF-35, desmin, cytokeratin, etc. The results were satisfactory similar to those by the ordinary methods and the time required was greatly shortened. Besides, the titres of antibody required were also similar as those used for routine immunohistochemical technique. It is assumed that application of microwaves in staining process might be valuable for pathological diagnosis and particularly the efficiency could be greatly enhanced.