Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor.

Stem cell factor (SCF) acts in concert with lineage-specific growth factors to stimulate the growth of hematopoietic colonies. To determine if neoplastic human hematopoietic cells would also respond to SCF, we cultured marrow mononuclear cells from 20 patients with newly diagnosed acute myelogenous leukemia (AML) and two normal donors with SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations of growth factors in semisolid medium, and assessed colony growth. SCF receptors (c-kit receptors) were quantitated by equilibrium binding studies with 125I-SCF, and binding parameters were estimated using the ligand program. The cellular distribution of c-kit receptors was determined by autoradiography. Our results show that SCF alone or in combination with IL-3 or GM-CSF increased both the number and size of colonies in 10 of the patients. Receptors for SCF were identified on the blasts from all 20 AML patients. The number of receptors ranged from 600 to 29,000 per cell. In the majority of patients, both high- and low-affinity binding sites were identified. Neither the number of receptors per cell nor the finding of one or two classes of receptors correlated with growth response to SCF. Autoradiographic analysis of 125I-SCF binding to normal marrow mononuclear cells revealed grains associated with blasts and megakaryocytes. Grain counts on blasts from 10 AML patients and on normal marrow blasts suggested that high-affinity c-kit receptor expression on AML blasts is lower than or similar to that of normal blasts. These results identify c-kit receptors on human AML blasts, and indicate that SCF acts synergistically with IL-3 or GM-CSF to stimulate colony growth from the marrow cells of a portion of patients with AML.


Hematopoietic stem cell depletion by restorative growth factor regimens during repeated high-dose cyclophosphamide therapy.

We studied the effects of six cycles of repeated cyclophosphamide (CTX) therapy followed by restorative therapy with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF on the hematopoietic stem cell compartment. Stem cell function was assessed by serially transferring bone marrow cells from CTX-CSF-treated mice into lethally irradiated recipient mice. Bone marrow cells from mice that initially received either G-CSF or GM-CSF after CTX therapy more rapidly lost the ability to repopulate the recipient lymphoid organs, showed a dramatic loss of hematopoietic progenitors, a more rapid loss of CFU-S capacity, and a 40% to 50% reduction in marrow repopulating ability (MRA). Interleukin-1 (IL-1) appeared to have little effect on the CTX-treated mice when used alone. However, when administered before the CTX-CSF regimen, IL-1 prevented the stem cell depletion as determined by CFU-C, CFU-S, and MRA through the serial transplantation procedures. These results support the hypothesis that repeated treatments with myelosuppressive drugs followed by stimulation with the CSFs may induce damage to the host stem cell compartment, and further suggest that pretreatment with IL-1 before CTX therapy may prevent this stem cell damage.


Functional reconstitution of the human interleukin-3 receptor.

The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL-5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL-2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3-responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.


Effect of stem cell factor with and without granulocyte colony-stimulating factor on neonatal hematopoiesis: in vivo induction of newborn myelopoiesis and reduction of mortality during experimental group B streptococcal sepsis.

Neonatal hematopoiesis and host defense are developmentally immature and under states of increased demand predispose the newborn to peripheral cytopenias and depletion of bone marrow storage pool reserves. We have previously demonstrated that recombinant human granulocyte colony-stimulating factor (rhG-CSF) can significantly modulate neonatal rat granulopoiesis and act synergistically with antibiotic therapy to reduce the mortality rate during experimental group B streptococcal sepsis. Stem cell factor (SCF) has been shown to stimulate early hematopoietic progenitor cells and, in the presence of lineage-specific CSFs, enhance committed progenitor cell proliferation. In the present study we examined the in vivo neonatal hematologic effects of recombinant rat (rr) SCF (14 days), simultaneous rrSCF + rhG-CSF (14 days), and sequential combination of rrSCF (7 days) + rhG-CSF (7 days). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneal (IP) x 14 days with the above combinations. rrSCF (0 to 200 micrograms/kg/d) had a negligible effect on the peripheral platelet count and absolute neutrophil count (ANC) but the diminution in the hematocrit during the first 10 days of treatment was less pronounced (P = .0001). However, the simultaneous use of rrSCF + rhG-CSF synergistically increased the circulating day 6 to 13 ANC (P = .001). Similarly, sequential rrSCF + rhG-SCF also had a synergistic significant effect during the second week of therapy on the circulating ANC (P = .01). The bone marrow neutrophil storage and proliferative pools were also significantly increased in newborn rats treated with rrSCF + rhG-CSF versus rhG-CSF (P = .02). The bone marrow and liver/spleen CFU-GM pool was unchanged; however, the CFU-GM proliferative rates were significantly increased in the rrSCF + rhG-CSF group (P = .04). rrSCF also induced a significant increase in the bone marrow and liver/spleen mast cell pool (P = .002). Lastly, rrSCF x 14 days +/- rhG-CSF significantly reduced the mortality rate at 48 and 120 hours after experimental group B streptococcus sepsis (P = .03 and .05, respectively). These data suggest that combination SCF + G-CSF therapy compared with G-CSF alone significantly increases the neonatal rat peripheral neutrophil count, bone marrow myeloid pools and proliferative rates, and induces a reduction in the mortality rate during experimental bacterial sepsis. SCF therapy may have future potential applications in the modulation of human neonatal hematopoiesis and host defense.


Intermediate filaments in Sertoli cells.

Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre- and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone-dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycle-dependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cell-supporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad.


Comparison of alcian blue and ruthenium red effects on preservation of outer envelope ultrastructure in methanotrophic bacteria.

Alcian blue (AB) and ruthenium red (RR) effects on ultrastructural preservation of the bacterial cell envelope of methanotrophs are compared. A previous successful method with RR that enhanced preservation of outer envelope layers in two representative methanotroph species is applied to other genera and species of methanotropic bacteria. Alcian blue is substituted for RR in this en bloc protocol. The effect of AB on preservation of these layers is assessed at the ultrastructural level and compared to RR for all species examined. Further, comparison with freeze etch and a fixation in the absence of either RR or AB is made. Both RR and AB are found to aid preservation and help visualize additional components of the cell envelope which are lost or minimized in a standard fixation not employing these cationic reagents. For some species, images obtained are similar between RR and AB procedures and agree with images seen by freeze etch. For other species, AB preserves extended filamentous material that is partially condensed even with the use of RR. Thus, use of AB improves the preservation of outer envelope structure in these organisms equally or more effectively than use of RR.


Successful treatment of primary gastric non-Hodgkin's lymphoma with chemotherapy and radiotherapy.

A case of primary gastric lymphoma is reported. The lymphoma was of the diffusely infiltrating type extending from the supraangular region to the prepyloric region of the stomach. The patient refused gastrectomy, therefore combined treatment with BACOP chemotherapy and radiotherapy was carried out. Complete remission was achieved without any major complications. The patient has remained free from relapse for over 20 months.


No effect of growth hormone treatment on axonal transport of slow component a in diabetic and nondiabetic rats.

A decreased axonal transport of slow component a (SCa), i.e., neurofilaments, is an early event in experimental diabetes as well as hypothyroidism, and common to these metabolic derangements are decreased levels of serum insulin-like growth factor I (IGF-I). To evaluate the possible connection between these facts, we investigated the effect of growth hormone (GH), which stimulates IGF-I production, on axonal transport of SCa in diabetic and nondiabetic rats. Serum concentrations of IGF-I fell from about 1500 micrograms/L in controls to about 600 micrograms/L in diabetics. GH treatment (100 mu/rat twice daily) normalized IGF-I for the first week of diabetes, after which the level decreased to the level of the untreated diabetics. The SCa transport velocity was found to be decreased in the diabetic nerves as previously reported [0.91 +/- 0.07 = mm/day, n = 9; (mean +/- SD) versus 1.01 +/- 0.09 mm/day, n = 8, in controls, 2 p less than 0.05). No changes were seen for the GH-treated groups (1.03 +/- 0.06 mm/day, (n = 11) in GH-treated controls). The lack of effect of GH treatment can be due to blockage of IGF-I synthesis or the decreased level of thyroid hormone, triiodothyronine (T3), in the diabetic rats.


Close linkage of retinoic acid receptor genes with homeobox- and keratin-encoding genes on paralogous segments of mouse chromosomes 11 and 15.

Retinoic acid is essential for normal development and growth of structures such as head and limbs, and it can act as morphogen or teratogen. Retinoic acid induces expression of genes such as the homeobox genes and keratin type I and type II genes. Retinoic acid receptors are nuclear transcription factors that play a key role in retinoid physiology. As part of the characterization of retinoic acid receptor gene family, linkage of genes encoding the three receptors was determined by using interspecific backcross and recombinant inbred strain analysis of restriction fragment variants. Retinoic acid receptor alpha is located on mouse Chromosome (Chr) 11 near the homeobox-2 complex and the keratin type I gene complex, whereas retinoic acid receptor gamma is on mouse Chr 15 near the homeobox-3 complex and the keratin type II complex. Close genetic proximity of these functionally related genes may be significant. We confirmed assignment of retinoic acid receptor beta to the centromeric portion of Chr 14. These linkage assignments provide further evidence for duplicated segments in the mouse genome.


Induced reciprocal translocation in transgenic mice near sites of transgene integration.

Transgenic mice (JCP0 #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P0) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P0 rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1(14) translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14(1) derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P0 gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP0 mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P0 gene probes, but found no evidence for a direct involvement of the endogenous P0 gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage--possibly induced by the microinjection procedure--the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14)1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.


Species- and tissue-specific transcription of complex, highly repeated satellite-like Bsp elements in the fox genome.

We have studied the transcription of highly repeated satellite-like Bsp elements containing the potential promoter boxes for RNA polymerase III in the genomes of adult silver and arctic foxes. The Bsp repeat transcripts were abundant enough to be detected by Northern blot and semiquantitative dot blot hybridizations, and the majority were found in the nuclear RNA fraction from arctic fox kidneys. Weak hybridization signals were revealed with the cytoplasmic RNA preparation from silver fox kidneys and with the nuclear RNA fraction from arctic fox liver, and their intensity was intermediate with the total RNA from arctic fox brain. Taken together, the data suggest possible genomic interspersion of some Bsp repeats in these two representatives of Canidae. The observed species-and tissue-specificity of the transcription of Bsp repeats suggests that they may potentially accomplish regulatory functions in the fox genomes.


Protective activity of inhaled frusemide against immunological respiratory changes and mediator release in guinea-pigs.

The antianaphylactic activity of inhaled frusemide was studied in ovalbumin-sensitized guinea-pigs. The exposure of the animals to frusemide aerosol (1% solution for 20 min) attenuated the respiratory response to ovalbumin challenge (aerosol 1% solution) and was associated with a significant reduction of blood histamine (70%; P less than 0.01) and thromboxane-B2 (35%; P less than 0.01) compared to control animals. Similar results were obtained in isolated lungs perfused via the trachea excised from ovalbumin-sensitized guinea-pigs exposed to frusemide aerosol (1% solution for 20 min). In this series of experiments frusemide significantly prevented the increase in tracheal perfusion pressure (45%; P less than 0.01) and the concomitant release into the pulmonary effluent of both histamine (75%; P less than 0.01) and thromboxane-B2 (39%; P less than 0.01). In another series of experiments, frusemide (1 x 10(-4) M) significantly reduced the immune release of histamine from mast cells of ovalbumin-sensitized rats. The inhibitory activity of frusemide was in the same range of potency (66%; P less than 0.01) as that of disodium cromoglycate (1 x 10(-4) M). These data taken together indicate that frusemide when given by inhalation prevents histamine release secondary to antigen-antibody reaction.


Influence of nitrovasodilators on bovine pulmonary histamine release.

The organic nitrates and related nitrovasodilators are relaxants of vascular and airway smooth muscle. Very little information is currently available regarding the influence of nitrates and related nitrovasodilators on pulmonary autacoid release. This study examined the influence of glyceryl trinitrate, isosorbide dinitrate and sodium nitroprusside on histamine release from bovine lung mince. Spontaneous histamine release from bovine lung mince was not altered by 0.1 nM to 1 microM glyceryl trinitrate, isosorbide dinitrate or sodium nitroprusside. Glyceryl trinitrate, isosorbide dinitrate and sodium nitroprusside produced a concentration-dependent decrease in A23187 (10 microM) stimulated histamine release. Glyceryl trinitrate also inhibited histamine liberation following the addition of compound 48/80. Further studies indicated that the inhibitory action of glyceryl trinitrate was reversed by coincubation with the guanylate cyclase inhibitor, methylene blue (10 microM). These findings indicate that glyceryl trinitrate, sodium nitroprusside and isosorbide dinitrate inhibit non-immunologically stimulated pulmonary histamine release and suggest that alterations in guanylate cyclase activity may influence pulmonary histamine release.


[Osteocalcin in diffuse toxic goiter].

Bone metabolism was investigated in toxic goiter. The authors obtained evidence on high levels of osteocalcin which marks bone formation. The time of the thyroid function recovery during thyrostatic therapy and indices of phosphorus-calcium metabolism disagree indicating delayed normalization of the bone tissue metabolism. There is a correlation between the thyroid function, osteocalcin and oxyproline that indirectly confirms the action of thyroid hormones on the bone matrix; between osteocalcin levels and other indices of bone metabolism (serum alkaline phosphatase activity and oxyproline excretion).


Effect of aprotinin on neutrophil function after major vascular surgery.

High-dose aprotinin reduces blood loss and blood transfusion requirements during liver transplantation and cardiac and vascular surgery. The mechanism of the haemostatic effect of aprotinin is unclear. A general effect on the anti-inflammatory response may be involved. Because leucocyte activation is part of this process, white cell function was measured in patients undergoing aortic surgery who received high-dose aprotinin therapy (n = 10) and was compared with the results from controls who did not (n = 10). The test group received an intravenous bolus (2 x 10(6) kallikrein inhibitor units) of aprotinin after induction of anaesthesia followed by continuous infusion (0.5 x 10(6) kallikrein inhibitor units/h) until the end of the operation. Blood samples were obtained before operation, immediately after surgery, and 1 and 7 days after operation. Aprotinin maintained significantly better postoperative white cell function as measured by bipolar shape formation (P less than 0.001), unstimulated nitroblue tetrazolium (NBT) reduction (P less than 0.001) and chemotaxis (P less than 0.001). Endotoxin-stimulated NBT reduction was similar in both groups, indicating that neutrophils from treated individuals retained the capacity to respond to oxidative stimuli. Aortic surgery activates neutrophils in vivo, as reflected by impaired chemotaxis and increased superoxide production. Aprotinin protects the cells against this potentially deleterious effect without affecting their ability to respond when provoked. Whether this affects leucocyte interaction with coagulation pathways and contributes to the reduction in blood loss remains to be determined.


Pantomime, praxis, and aphasia.

The production and comprehension of pantomimed movements by asphasic subjects were studied with respect to their relationship to the aphasic deficit on one hand and apraxia on the other. At issue was whether impaired use of pantomime is a manifestation of reduced symbolic capacity or purely a manifestation of an apraxic impairment of purposeful movement or both. Tests of pantomime included a Pantomime Recognition Test, a nonverbal Transitive Pantomime Production Test, and an Intransitive Pantomime Production Test (to oral command). Imitation of Nonmeaningful Movements served as a measure of apraxia, uncontaminated by symbolic or linguistic factors. Imitation of Meaningful Movements taken from the pantomime tasks was also tested. Independent measures of auditory comprehension, picture naming, and reading comprehension were used as indices of language impairment. Intellectual function was measured by the Performance scale of the WAIS. Thirty aphasic subjects were examined. Twenty healthy age-matched normals served to establish scoring standards for the pantomime tests. Multiple regression analysis revealed significant, independent contributions from both auditory comprehension and imitation of Nonmeaningful Movements to all of the tests of pantomime production and pantomime recognition. All measures of pantomime production and pantomime recognition were strongly intercorrelated. While the three language measures were strongly correlated with each other, auditory comprehension was the only one of them that was significantly and consistently related to the pantomime tests. None was related to the imitation of nonmeaningful movement. The results are taken as indicating (1) that pantomime production and imitation share common factors with both praxis and auditory comprehension; (2) apraxia entails impairment of both production and interpretation of purposeful movements. Possible theoretical accounts for these results are offered.


Relation of linguistic communication abilities of Alzheimer's patients to stage of disease.

A battery of linguistic communication (L-C) tasks was administered to 152 Alzheimer's disease patients in different stages of the disease and 60 normal elders. Subject performance data are used to construct a profile of L-C deficits by disease stage, as determined by ratings on the Global Deterioration Scale. Specification also is made of the L-C tasks on which mild Alzheimer's patients perform like normal elders, the relative difficulty of various L-C processes, the disease stage in which the greatest change occurs in L-C functions, and the degree of variation in L-C for individuals at a particular level of dementia severity.


Organization of cutaneous primary afferent fibers projecting to the dorsal horn in the rat: WGA-HRP versus B-HRP.

Primary afferent projections from cutaneous afferents in the forelimb and hindlimb digits to the dorsal horn (DH) were examined using 4 tracers: (1) 25% free horseradish peroxidase (HRP), (2) 2.5% wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), (3) a mixture of 25% free HRP and 2.5% WGA-HRP (WGA-HRP/HRP) or (4) 0.1% HRP conjugated to cholera toxin (B-HRP). The tracer was injected intracutaneously into the digits. Three to 4 days later, the rats were perfused transcardially, transverse sections (60-microns thick) were cut and the HRP was reacted using the tetramethyl benzidine (TMB) method. The location of the label was reconstructed by camera lucida drawings. In rats which received an injection of HRP alone, no label was detected in the DH. Rats injected with WGA-HRP had projection patterns similar to those injected with WGA-HRP/HRP. Patterns of labelling with WGA-HRP differed markedly from those with B-HRP. WGA-HRP labelled cutaneous afferents projecting to Rexed's laminae I-III, with the densest label in lamina II; in contrast, B-HRP labelled cutaneous afferents projecting to laminae II-V, with the densest label in laminae III-IV. These results indicate that, for cutaneous primary afferents projecting to the DH, WGA-HRP and B-HRP labelled different subpopulations of fibers, with the B-HRP-labelled subpopulation biased toward afferents of larger diameter. Rostrocaudally, the extent of the densest fiber projections, whether labelled by WGA-HRP or by B-HRP, was essentially the same, but the extent of the less densely labelled projections was much greater with B-HRP than with WGA-HRP. Comparisons of the projection maps from each of the five digits, using either WGA-HRP or B-HRP, indicated that, as seen in transverse sections through the DH, there was extensive overlapping among the labelled cutaneous afferent fibers from adjacent, or even non-adjacent digits.


Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines.

Acidic fibroblast growth factor (aFGF) enhances nerve growth factor (NGF) synthesis by astrocytes obtained from various brain regions. NGF secretion by fibrous-shaped astrocytes transformed by dibutyryl-cAMP (db-cAMP) pretreatment was less than that by untreated astrocytes. However, aFGF also enhanced NGF secretion by fibrous-shaped astrocytes. The effects of various kinds of intracellular signaling modulators on NGF synthesis were examined. None of the following second messenger effectors had an effect on NGF synthesis: protein kinase C (PKC) agonist (phorbol myristate acetate (PMA)) or antagonist (sphingosine (SP)). LiCl, and ionomycin (Iono). Further, increases of intracellular cAMP by forskolin (FK) or db-cAMP have no significant effect on NGF synthesis in astrocytes under a standard culture condition. However, NGF synthesis by astrocytes in the presence of aFGF was significantly enhanced by db-cAMP, but not by FK or sodium butyrate. These results indicate that an excessive amount of cAMP enhances the effect of aFGF on NGF synthesis in astrocytes. NGF synthesis in astrocytes was not affected by treatment with anti-aFGF or anti-bFGF neutralizing antibodies, indicating that FGFs are not involved in the autocrine regulation of NGF synthesis in astrocytes. Transforming growth factor-beta 1 (TGF-beta 1), which inhibits some effects of FGFs, increased NGF synthesis in concert with aFGF. Furthermore, the highest NGF synthesis was observed when astrocytes were stimulated by all of the following cytokines: aFGF, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and TGF-beta 1. The mechanism regulating NGF synthesis in fibroblasts obtained from prenatal rat skin was also investigated. Acidic FGF, basic FGF (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), TGF-beta 1, IL-1 beta, and TNF-alpha were found to be regulators of NGF synthesis in skin fibroblasts. Among these cytokines, aFGF is the most potent regulator of NGF synthesis in fibroblasts. NGF synthesis by skin fibroblasts, either in the presence or absence of aFGF, was not modified by any of the following: FK, PMA, SP, LiCl, and Iono. However, db-cAMP significantly enhanced NGF synthesis in both conditions. Sodium butyrate enhanced NGF synthesis in the presence of aFGF, but not in the absence of aFGF. These results suggest that an excessive amount of cAMP and butyrate moiety regulate NGF synthesis in skin fibroblasts in different ways.(ABSTRACT TRUNCATED AT 400 WORDS)


A second auditory area in the non-cortical telencephalon of a reptile.

Injections of horseradish peroxidase (HRP) into a caudocentral portion of the non-cortical telencephalon of Caiman known as the dorsolateral area (dorsal ventricular ridge) resulted in retrogradely labeled neurons throughout the entire extent of the ipsilateral nucleus reuniens. HRP-positive cells were most numerous in nucleus reuniens pars diffusa with only sparse labeling of neurons in nucleus reuniens pars centralis. The results of the present experiment, when compared with those of a prior study that determined the telencephalic connections of nucleus reuniens pars centralis, suggested that these two forebrain areas are separate. Staining with succinate dehydrogenase and acetylcholinesterase revealed that nucleus reuniens pars centralis and pars diffusa and their respective telencephalic projection areas can be differentiated on the basis of histochemical features. These findings in Caiman suggest that certain thalamic and telencephalic auditory areas in birds and crocodilians are most likely the result of common ancestry rather than examples of parallel evolution.


Effects of intravenous infusion of substance P on arginine vasopressin and oxytocin secretion in normal men.

In order to establish possible stimulatory effects of increasing plasma concentrations of substance P (SP) on the arginine vasopressin (AVP) and/or oxytocin (OT) secretion, successively increasing doses of SP(0.5, 1 and 1.5 pmol/kg-1/min-1; each dose for 20 min) were infused in 7 normal men. Plasma AVP and OT levels were measured before infusion and every 20 min, just before increasing the infusion dose of SP. During tests, SP infusion did not produce untoward side effects or changes in blood osmolality and/or pressure. Plasma OT levels did not change during SP infusion. Plasma AVP concentrations were not modified by the infusion of the lowest dose of SP, whereas they were significantly increased in a dose response fashion when higher amounts of SP were given. These findings demonstrate for the first time in humans that the systemic administration of SP exerts stimulatory effects on AVP, but not on OT secretion.


A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies.

The monoclonal antibody 62-3G1 raised against the GABAA/benzodiazepine receptor complex was tested for its subunit selectivity using recombinantly expressed GABAA receptor subunits. The antibody bound selectively to beta 2 and beta 3 but not beta 1 nor any other GABAA receptor subunit. Using enzyme-linked immunosorbent assay, the epitope on beta 2 and beta 3 subunits was determined to be residues 1-3. MAb bd 17, which displays identical subunit selectivity as mAb 62-3G1, was seen to bind to the same epitope. These results resolve the subunit selectivity of mAbs 62-3G1 and bd 17 and reveal the identity and localization of a prominent immunogenic epitope on the GABAA/benzodiazepine receptor.


Immunocytochemical localization of endopeptidase 24.15 in rat brain.

Endopeptidase 24.15 (EC 3.4.24.15; EP 24.15), a zinc-metalloendopeptidase highly active in rat testes, brain and pituitary, converts some prodynorphin- and proenkephalin-derived oligopeptides into the corresponding enkephalins and degrades a variety of bioactive peptides including bradykinin, neurotensin, and both angiotensin I and II. The immunocytochemical localization of the enzyme was studied in rat brain using a polyclonal antibody raised in rabbits against a homogeneous preparation of the enzyme isolated from rat testes. The distribution of EP 24.15 immunoreactivity in the brain was widespread, being present in both neurons and glial cells. Surprisingly, however, staining was predominantly nuclear, and not cytoplasmic as expected based on the biochemical demonstration that EP 24.15 activity is predominantly associated with the soluble protein fraction of brain homogenates. Cytoplasmic staining was detected in large neurons but was less intense than the nuclear staining. The highest density of EP 24.15-staining was detected in nuclei of cerebellar Purkinje cells and in hippocampal dentate gyrus cells. High levels of immunoreactivity were also noted in brain areas which contain peptides known to be substrates of the enzyme in vitro. This localization supports a role for EP 24.15 in neuropeptide metabolism, but also suggests an as yet undefined role in nuclear function.


GABA-like immunoreactive neurons in the retina of Bufo marinus: evidence for the presence of GABA-containing ganglion cells.

gamma-Aminobutyric acid (GABA)-like immunoreactive (IR) neurons in the retina of the cane toad Bufo marinus were revealed using immunohistochemistry on retinal wholemount preparation and sectioned material. GABA-IR neurons included horizontal, bipolar and amacrine cells in the inner nuclear layer and small to medium sized cells in the ganglion cell layer. A few IR axons were seen in the optic fiber layer of the retina. Following the injection of the carbocyanine dye, DiI into the optic tectum ganglion cells were retrogradely filled. A small population of DiI-filled ganglion cells (2.8%) was found to be GABA-IR. GABA-IR neurons in the ganglion cell layer without DiI label were considered to be displaced amacrine cells of which 45.3% were GABA positive. It is proposed that GABA-containing ganglion cells may form an inhibitory projection to visual centers of the anuran brain.


Expression of granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor genes in human astrocytoma cell lines and in glioma specimens.

Expression of granulocyte (G) and granulocyte-macrophage (GM) colony stimulating factor (CSF) genes in human cells of astroglial lineage was studied. Primers for CSFs were used to analyze RNA transcripts in 5 cultured human astrocytoma cell lines and 8 fresh brain specimens by polymerase chain reaction. Constitutive expression of mRNA transcripts of GM-CSF could be detected in all astrocytoma and one neuroblastoma cell lines, and two out of 5 unstimulated astrocytomas, U87MG and U138 MG, expressed G-CSF genes. After stimulation with interleukin (IL)-1 beta + tumor necrosis factor (TNF)-alpha, all cell lines expressed G-CSF. In addition to the cultured cells, we examined gene expression within human malignant astrocytoma, peritumoral brain and autopsied normal brains. The results show that some of the tumor and its surrounding reactive lesions express G- and GM-CSF genes but normal brains do not. The concentration of G- and GM-CSF in supernatants of cultured cells was assessed at the protein level by ELISA. A low level of GM-CSF activity was constitutively present in all astrocytomas. G-CSF was detected in unstimulated U87MG and U138MG and other cell lines could synthesize G-CSF after the stimulation of IL-1 beta and TNF-alpha at the level of mRNA. Furthermore, the concentration of CSFs increased markedly upon stimulation with IL-1 beta and/or TNF-alpha in both a time- and dose-dependent fashion. From these results, it is suspected that astroglial cell-derived CSFs may participate in local immune reactions accompanying infection, degeneration and malignancies in the brain.


Glutamic acid-insensitive [3H]kainic acid binding in goldfish brain.

Kainic acid is supposed to be a specific agonist for a subclass of excitatory glutamate receptors in the vertebrate CNS. An investigation of (2 nM) [3H]kainic acid binding sites in goldfish brain, using quantitative autoradiography, has revealed evidence for two types of kainic acid receptors which differ in sensitivity to glutamic acid. L-Glutamic acid (0.1-1 mM) displaced over 95% of specific [3H]kainic acid binding elsewhere in the brain but only 10-50% in the cerebellum and cerebellar crest. These structures apparently contain [3H]kainic acid binding sites that are extremely insensitive to glutamic acid. The glutamic acid-insensitive [3H]kainic acid binding was not displaced by quisqualic acid, kynurenic acid, alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA), or N-methyl-D-aspartatic acid, but was completely displaced by the kainic acid analogue domoic acid. The data indicate that two types of high affinity binding sites for [3H]kainic acid exist in the goldfish brain: glutamic acid-sensitive and glutamic acid-insensitive. High affinity [3H]kainic acid binding may therefore not always represent binding to subsets of glutamic acid receptors.


Mechanisms of action of ibogaine and harmaline congeners based on radioligand binding studies.

Assays using radioligands were used to assess the actions of ibogaine and harmaline on various receptor types. Ibogaine congeners showed affinity for opiate receptors whereas harmaline and harmine did not. The Ki for coronaridine was 2.0 microM at mu-opiate receptors. The Kis for coronaridine and tabernanthine at the delta-opiate receptors were 8.1 and 3.1 microM, respectively. Ibogaine, ibogamine, coronaridine and tabernanthine had Ki values of 2.08, 2.6, 4.3 and 0.15 microM, respectively, for kappa-opiate receptors. Long-lasting, dose-dependent behavioral effects of ibogaine have been reported. The possibility that these effects were due to irreversible binding properties of ibogaine at kappa-receptors was considered; however, radioligand wash experiments showed a rapid recovery of radioligand binding after one wash. A voltage-dependent sodium channel radioligand demonstrated Ki values in the microM range for all drugs tested. Using radioligand binding assays and/or 36Cl- uptake studies, no interaction of ibogaine or harmaline with the GABA receptor-ionophore was found. The kappa-activity of ibogaine (or an active metabolite) may be responsible for its putative anti-addictive properties whereas the tremorigenic properties of ibogaine and harmaline may be due to their effects on sodium channels.


Recovery from lateralized neocortical damage: dissociation between amphetamine-induced asymmetry in behavior and striatal dopamine neurotransmission in vivo.

It has been hypothesized that neocortical damage is accompanied by secondary changes in other brain areas (the shock or diaschisis of von Monakow), which contributes to initial non-specific behavioral depression. The relation between behavioral changes and dopamine (DA), serotonin (5-HT), and their metabolites, measured with intracerebral microdialysis in freely moving rats and by tissue assay postmortem, was examined during postsurgical recovery from unilateral hemidecortications. Rats were tested for rotational asymmetry and extracellular concentration of DA was measured both during rest and after amphetamine (1.5 mg/kg). It was found that: (1) during the first few postsurgical days the hemidecorticate rats rotated ipsilateral to their lesions after amphetamine but thereafter on tests given up to 121 days postsurgery concentration of DA or its metabolites at any time after surgery; (3) the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) was elevated acutely for a few days following surgery; (4) during the first 3 postoperative days, both baseline extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) and amphetamine-induced DA release were significantly elevated bilaterally. These findings demonstrate that the acute behavioral asymmetry in rotation produced by hemidecortication is not related to unilateral changes in striatal DA activity and its metabolites. Thus, the behavioral asymmetries might be related to other striatal changes (i.e. 5-HIAA) or other damage, such as to the corticospinal projections of the lesioned hemisphere. Nevertheless, unilateral lesions did produce acute bilateral increases in DA levels, which may be a correlate of generalized neural shock produced by the lesion.


In vivo microdialysis reveals a diminished amphetamine-induced DA response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment.

In vivo microdialysis procedures were used to assess the effects of repeated amphetamine administration on behavior and regional brain DA dynamics in freely moving rats. Pretreatment with amphetamine (2.5 or 3.0 mg/kg) for 4-6 days did not alter baseline DA or its metabolites in caudate or accumbens 48 h or 6 days after the last injection. However, whereas this dosage regimen revealed a profound behavioral sensitization in response to challenge with amphetamine (2.5 mg/kg), including a more rapid onset and intensification of stereotypy, the DA response was significantly diminished in both brain regions. In addition, the ratio of caudate to accumbens DA, either before or after amphetamine challenge, was not altered by the pretreatment regimen. These results are consistent with our previous suggestion that there is a dissociation between the DA and behavioral responses to amphetamine, and therefore that other neurotransmitter systems and/or mechanisms significantly contribute to the amphetamine response profile. Furthermore, DA effects may represent only one, albeit critical, aspect in a time-dependent sequence of changes underlying stimulant sensitization.


Ethanol inhibition of N-methyl-D-aspartate-activated ion current in rat hippocampal neurons is not competitive with glycine.

The interaction of ethanol with glycine at the N-methyl-D-aspartate (NMDA)-activated ion channel was investigated in voltage-clamped rat cultured hippocampal neurons. As shown previously, glycine increased, and ethanol inhibited, the NMDA-activated current in these cells. Concentration-response data for glycine (0.1-100 microM) indicate that the inhibition of NMDA-activated current by ethanol does not involve a competitive interaction with glycine. Thus, ethanol appears to inhibit NMDA-activated current at a locus different from the glycine modulatory site.


Ultrastructural double-labeling demonstrates synaptic contacts between dopaminergic terminals and substance P-containing striatal neurons in pigeons.

Immunohistochemical studies in rats have demonstrated dopaminergic input onto medium spiny neurons of the striatum. Medium spiny neurons, however, are known to consist of two major neuropeptide-specific types, those containing substance P (SP) and those containing enkephalin. Although both of these types have been shown to receive dopaminergic input onto their perikarya and proximal dendrites, the extent to which both types also receive direct dopaminergic input onto distal dendritic shafts or onto dendritic spines is uncertain. In the present study, we used EM immunohistochemical double-label techniques to examine the synaptic organization of dopaminergic input onto SP+ striatal neurons. We examined the striatum of pigeons, in whom SP+ striatal neurons, including their dendritic shafts and spines, can be readily labeled. Antibodies against tyrosine hydroxylase (TH) were used to identify dopaminergic terminals, which were labeled using silver-intensified immunogold. The SP+ neurons were labeled immunohistochemically using diaminobenzidine. We found that dopaminergic terminals make appositions and form symmetric synapses with the perikarya, dendritic shafts and dendritic spines of SP+ neurons. Thus, nigral dopaminergic neurons provide a monosynaptic input onto SP+ striatal neurons in a manner similar to that described for dopaminergic input onto striatal medium spiny neurons in general.


Inhibition of proliferative responses of lymphocytes to food antigens by an anti-allergic drug, N(3',4'-dimethoxycinnamoyl) anthranilic acid (Tranilast) in children with atopic dermatitis.

Experimental studies have shown that N(3',4'-dimethoxycinnamoyl) anthranilic acid (Tranilast) inhibits reaginic antibody-mediated hypersensitivity reactions, and it has been demonstrated to be an effective drug for patients with bronchial asthma. On the other hand, from the nature of the cellular infiltrate seen in eczematous lesions, it appears that some form of cell-mediated immunity may be involved in addition to IgE-mediated immunity in the pathogenesis of atopic dermatitis (AD). Moreover, we have previously reported that the proliferative responses of peripheral blood mononuclear cells (PBMCs) to ovalbumin (OA) or bovine serum albumin (BSA) in children with AD who are sensitive to hen's egg or cow's milk were significantly higher than those of healthy children and hen's egg or cow's milk sensitive children with immediate symptoms. In this study, we have showed that the proliferative responses of PBMCs to OA were dose-dependently inhibited by Tranilast on patients with AD. The responding cells to OA were shown, through separation experiments, to be T cells, and the proliferative responses of T cells to OA were also dose-dependently inhibited by Tranilast. Moreover, the inhibition was thought to occur at the initial stage of the proliferative reactions. These results suggest that Tranilast can be clinically applied to patients with AD.


Crossreacting IgE antibodies to Pityrosporum ovale and Candida albicans in atopic children.

Sera from 13 patients with positive Pityrosporum ovale skin prick test were analysed with IgE immunoblotting. Both P. ovale and Candida albicans antigens were used to reveal possible crossreactivity of these yeasts. Of the 13 sera, eight sera showed IgE binding to P. ovale and six sera to C. albicans. Simultaneous IgE-binding to both C. albicans and P. ovale was observed in five of these sera. The most common IgE-binding band pairs were, a 23 kD band of P. ovale and a 27 kD band of C. albicans, as one pair, and a 26 kD band and a 13 kD band, respectively, as another pair. In addition to these protein bands, simultaneous reactivity was observed to C. albicans mannan, a polysaccharide, and to a diffuse high molecular weight component of P. ovale, possibly also a polysaccharide. The five sera with simultaneous IgE-binding were analysed with RAST-inhibition, which revealed presence of crossreacting IgE-antibodies to P. ovale and C. albicans in two of these sera. Crossreacting epitopes were suggested to be located in the mannan polysaccharide of C. albicans and high molecular weight diffuse stain of P. ovale, based on the immunoblotting and RAST-inhibition patterns of the studied sera. Crossreacting sera were pooled and used as an IgE probe in CRIE and Tandem-CRIE. These experiments revealed a fused precipitin line indicating presence of a common structure of P. ovale and C. albicans. This suggested crossreactivity may imply an interesting phenomenon in atopics who are sensitized by C. albicans growth in the gastrointestinal tract and get exposed by P. ovale growing on the skin.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum.

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of Penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two-dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS-PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.


Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.


Child psychiatry and early intervention: III. The developmental disorders.

This paper is the third in a series of four using the concepts introduced in the paper Child Psychiatry and Early Intervention: I. The Aggregate Burden of Suffering. This paper reviews the developmental disorders of childhood to set priorities for early intervention programs. This review discusses the prevalence, course, risk, early indicators, associated impairments, and responses to intervention. The specific developmental disorders, especially learning disabilities, have a significant impact on community resources. Since the developmental disorders are easily recognized among preschool children, early intervention is feasible. However, successful interventions have yet to be demonstrated. Priority should be given to the development of effective interventions. There is a great need for research studies on the effects and effectiveness of early intervention with these individuals.


Amiloride but not bumetanide protects against the cytotoxic effects of estramustine and bleomycin in cultured fibroblasts.

The effects of the clinically used diuretics amiloride (an inhibitor of Na+/H+ exchange) and bumetanide (an inhibitor of Na+, K+, Cl- co-transport) were tested on the cytotoxicity of estramustine and bleomycin in cultured fibroblasts. Both estramustine (50 micrograms/ml) and bleomycin (10 micrograms/ml) reduced the number of surviving clones. Amiloride (100 microM) but not bumetanide (100 microM) partly protected against the cytotoxic effect of both estramustine and bleomycin. The protective effect of the combination of amiloride and bumetanide was not stronger than the effect of amiloride alone. Amiloride or bumetanide alone did not affect the clonal survival. It is suggested that the protective effect of amiloride is not mediated by an effect of the molecular mechanism of cytotoxicity but may rather be due to changes in cellular metabolism and/or drug handling.


A nuclear mutation affects the synthesis of the chloroplast psbA gene production Chlamydomonas reinhardtii.

The effect of the nuclear mutation F34 on the synthesis of chloroplast-encoded photosystem II (PSII) polypeptides has been controversial. While we had concluded that the synthesis of the psbC gene product (P6) was specifically deficient in this mutant, another laboratory has found that the synthesis of the psbA gene product, the herbicide-binding protein D1, was primarily affected. These conflicting results were re-analyzed through genetic and biochemical characterization of the different strains used by the two laboratories in question. While the strain used in our laboratory carries the single F34 mutation responsible for the deficiency of P6, the other strain contains three nuclear mutations: the original F34 mutation, a suppressor mutation of F34 and a second PSII mutation, F35. Mutation F35 does not affect P6 synthesis but is responsible for the deficiency in the synthesis of the herbicide-binding protein D1. Furthermore, since both F34 and F35 mutant strains accumulate wild-type levels of psbC and psbA transcripts, we show that these nuclear mutations affect nuclear genes whose products are involved in the expression of psbC or psbA at a post-transcriptional level.


The higher plant nad5 mitochondrial gene: a conserved discontinuous transcription pattern.

The single copy nad5 gene has been identified in the mitochondrial genomes of cauliflower, chicory, potato, fennel, and common bean. In these five dicot species the same organization as in Oenothera, Arabidopsis, wheat, and maize has been found for the gene: it consists of five exons organized into three independent groups. The first group comprises exons I and II, separated by a highly conserved group II intron, while the second group consists of exon III only. In the third group exons IV and V are separated by a group II intron of variable, species-specific, length. Transcription analysis of the nad5 gene in chicory and in cauliflower shows that the five exons are assembled as a mature mRNA through intermolecular interactions and multiple splicing events. Comparison of transcription in the gene with that of wheat and maize suggests that a common mechanism exists in higher plants for nad5 transcript processing.


Chemical modification of fumagillin. II. 6-Amino-6-deoxyfumagillol and its derivatives.

6-Amino-6-deoxyfumagillol (5) was synthesized by reductive amination of 6-oxo-6-deoxyfumagillol (4), which was obtained by oxidation of fumagillol (2). The reduction proceeded stereoselectively by the equatorial attack of hydride and 5 was found to have the same stereochemistry as that of 2. Several derivatives of 5 were prepared and most of them showed anti-angiogenic activity comparable to that of fumagillol derivatives.


Suppressive effect of polyoxometalates on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and their inhibitory activity against HIV-1 reverse transcriptase.

One isopolyoxometalate and 42 heteropolyoxometalates consisting of 3 compounds with the trivacant Keggin structure, 2 with the lacunary Keggin structure, 30 with the Keggin structure, one with the Wells-Dawson structure and 6 with miscellaneous structures were tested for their suppressive effect on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and inhibitory activity against HIV-1 reverse transcriptase. In contrast to the leading interpretations which attribute the suppressive effect of polyoxometalates on the cytopathogenicity of HIV-1 to the inhibition of HIV-1 reverse transcriptase by these compounds, there was no distinct correlation observed between these two functions of polyoxometalates.


Treatment of the carcinoid syndrome with somatostatin, salmon calcitonin, or octreotide.

Three patients with the carcinoid syndrome received intravenous somatostatin (3.5 micrograms/min) for one day; intravenous salmon calcitonin (8 IU/hr) for one day; subcutaneous salmon calcitonin (100 IU three times daily) for ten days; and subcutaneous octreotide (150 micrograms three times daily) for ten days. Octreotide (SMS-201.995) is a stable analogue of somatostatin. There was a five-day washout period between each treatment. During each of these treatments, reductions in the numbers of daily flushes and bowel movements, stool weight, and urinary 5-hydroxyindoleacetic acid (5-HIAA) levels were observed. Relief of cramping abdominal pains was also reported. Patients 1 and 3 chose to continue receiving the subcutaneous calcitonin and patient 2 chose the octreotide. Patient 1 (aged 67 years) reported relief of symptoms for five months until she developed an intestinal obstruction as a result of tumor infiltration. Patient 3 (aged 67 years) has received the calcitonin for about 16 months with relief of symptoms and reduced urinary 5-HIAA levels. Patient 2 (aged 57 years) has continued octreotide treatment for one year and reports relief of symptoms.


Synergistic antiproliferative effect of interferons and azidothymidine on HL60 cells.

Proliferation of human leukemia cells, HL60, was synergistically inhibited by a combination of human interferons and azidothymidine in vitro. Combination of interferon-gamma (3000 U/ml) and azidothymidine (30 microM) inhibited cell growth by 76%, whereas interferon-gamma alone suppressed growth by 23% and azidothymidine alone by 33%. Interferon-alpha-2a and interferon-beta also exerted synergistic effects with azidothymidine, but the potentiation was weaker than that by the combination of interferon-gamma and aziodthymidine.


Reduction of endotoxin-induced vascular permeability by monoclonal antibodies against lipopolysaccharide determinants.

Endotoxin, a bacterial lipopolysaccharide implicated in the pathogenesis of septic shock, markedly alters vascular permeability following intravenous injection in rabbits. We investigated the ability of murine monoclonal antibodies to confer protection against endotoxin-induced increases in a rabbit model of ocular vascular permeability. Four monoclonal antibodies of differing specificities as well as polymyxin B were compared for their effects on endotoxin from either Escherichia coli or Pseudomonas aeruginosa. Preincubation of endotoxin with antibodies directed against Pseudomonas O side chain or core glycolipid resulted in marked attenuation of vascular permeability due to Pseudomonas endotoxin, but not E. coli endotoxin. Antilipid A antibodies were not significantly effective in neutralizing either endotoxin with in vitro preincubation. Low avidity of the antilipid A antibody, low density of lipid A binding sites, or inaccessibility of the lipid A may have prevented more marked interactions. When administered intravenously prior to endotoxin challenge, none of the antibodies demonstrated the ability to provide specific protection to subsequent endotoxin in this model. They did provide partial nonspecific protection against endotoxins regardless of epitope specificity. When administered prophylactically, polymyxin B, an antibiotic that binds to lipid A, was highly effective in neutralizing the toxic effects of endotoxin. Since antibodies to lipid A reduce mortality in septic shock, the failure to demonstrate efficacy in this study may be due to the marked sensitivity of the rabbit eye to endotoxin. Alternatively, beneficial effects from antiendotoxin antibodies in septic shock may be unrelated to the inhibition of vascular permeability. Some protection from antiendotoxin antibodies may be due to enhancement of nonspecific mechanisms.


Development of aging-associated monoclonal gammapathies with antibody activity to the antigen used for immunization of young mice.

The influence of immunization with dinitrophenylated human serum albumin (DNP-HSA) at a young age on the development of age-related monoclonal gammapathies (MG) was investigated in a longitudinal study in intact and neonatally thymectomized (NTx) C57BL/KaLwRij and CBA/BrARij mice. Three-month-old mice were immunized four times in monthly intervals with DNP-HSA. Control mice received saline and adjuvant only. Mice immunized with DNP-HSA responded with heterogeneous antibodies, occasionally with some clonal dominance. The antibody levels further declined and were hardly detectable when the mice were 21 months old. Eighteen of 87 experimental mice developed homogeneous antibody components (H-Ab) to DNP-HSA with aging. Their frequencies per individual groups were 5, 22, 24, and 29% for intact CBA, NTx-CBA, NTx-C57BL, and intact C57BL mice, respectively. Some H-Ab had the same mobility and similar spectrotypes as dominant clonal products at the peak of the response. However, the majority of H-Ab appearing at old age were "new" H-Ab. While most of H-Ab in the CBA mice were transient and of a low concentration, the majority of H-Ab in the C57BL mice had all characteristics of a benign monoclonal gammapathy. The results indicate that memory cells of the B cell clones involved in the original specific response may in a susceptible strain become targets for events leading to the development of benign monoclonal gammapathy.


Shift of private and not of cross-reactive anti-DNA idiotypes in systemic lupus erythematosus.

The shift of private idiotype (Id) and cross-reactive Id (CRI) on anti-DNA antibodies in a lupus patient KE was investigated during a 7-year period. Anti-private Id and anti-CRI activities were separated by affinity chromatography from rabbit (R)-anti-Ids raised against KE anti-DNA antibodies during active (1/84) and inactive (4/90) stages of the disease. Anti-CRI isolated from the 84 R-anti-Id appeared to recognize binding site-related Ids that are shared with KE non-anti-DNA antibodies, unrelated lupus patients' sera, and certain normal sera. Id expression on serial serum samples of KE using these fractionated R-anti-Ids as probes showed that the 1/84 private Id expression declined while the 4/90 private Id expression gradually increased. Expression of the CRI showed a relatively stable pattern. These results suggest that anti-DNA populations detected by anti-private Id can shift, while populations expressing CRI may stay stable.


[A case of acute idiopathic pandysautonomia--a histochemical study of sural nerve by acetylcholinesterase staining].

A 30-year-old man had an acute onset of orthostatic lightheadedness, sweating disturbance, paroxysmal cough and loss of potency. These symptoms reached the peak in two weeks, and then remitted very slowly. He was admitted to our hospital for further evaluation when he was 39 years old. Neurological examinations revealed right Horner's syndrome, dry skin and impotence, but neither motor nor sensory system was impaired. No abnormalities were found on routine examinations of the blood and cerebrospinal fluid, motor and sensory nerve velocities, computed tomography and electroencephalography. On sural nerve biopsy, the density of unmyelinated fibers was mildly decreased (13,857/mm2), whereas that of myelinated fibers was normal (7,220/mm2). Autonomic function tests disclosed orthostatic hypotension (-31 mmHg) on tilting, reduced levels of serum noradrenaline and vanillyl mandelic acid, supersensitive responses to noradrenaline infusion and adrenaline eye-dripping, severe sweating impairment and complete absence of sympathetic skin response. On the other hand, Aschner's test, Czermak's test and coefficient variation of R-R intervals were all normal. These results suggested that the chief lesion was located in the postganglionic fiber of sympathetic efferent pathway. We (Hayashi et al, 1990) quantified acetylcholinesterase (AchE)-positive fibers in the specimens of sural nerve biopsy, and reported that the density of AchE-positive fibers was correlated to the function of sympathetic postganglionic fibers. The density of AchE-positive fibers in the present case of acute idiopathic pandysautonomia (AIPD) was severely decreased to 225/mm2 by optical microscopy (control: 5,703 +/- 1,289/mm2), and to 2,996/mm2 by electron microscopy (control: 14,112 +/- 3,987/mm2).(ABSTRACT TRUNCATED AT 250 WORDS)


Biochemical parameters as a measure of food availability and growth in immature rainbow trout (Oncorhynchus mykiss).

1. Rainbow trout held in brackish water (15 parts per thousand) were starved or fed different amounts of food. 2. A significant correlation was found between the growth rates of the different animals and the feed rates. 3. The RNA:DNA ratio in the white epaxial muscle is lowest in starved fish and increases in proportion to the feed rate and individual specific growth rate. The correlations are significant at the P less than 0.01 level. 4. Liver metabolism varies according to food availability. 5. The protein synthesis capacity of the liver (RNA:DNA ratio) and liver somatic index increase as the feeding rate increases. It also correlates significantly with the specific growth rates of the different animals. 6. The intermediary metabolism of the central metabolic organ, the liver, varies in the same way. 7. The activities of the NADPH producing liver enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (IDH) and malic enzyme (ME) increase as the feed rate (and therefore the specific growth rate) increases. 8. G6PDH and IDH activity in the kidney is influenced to a much lower degree by food intake. 9. Summarizing, it can be stated that biochemical parameters can be used to describe comprehensively the metabolic status and growth of rainbow trout.


Adaptive significance of amylase polymorphism in Drosophila--VI. Properties of two amylase variants and the effect of food components on amylase activity in Drosophila subobscura.

1. Properties of amylase from two D. subobscura strains homozygous for two different amylase variants (AmyS and AmyF) were determined. 2. Amylase of both strain adults showed a pH optimum of 7.8. 3. The AmyF enzyme showed a higher thermostability. 4. They differed in both maximum activity and Michaelis constant (Vmax of 6.25 and 3.45, Km of 0.7% and 0.42% starch for AmyS and AmyF, respectively). 5. The effect of different feeding conditions in amylase activity in the above Drosophila strains was also studied. Amylase activity was always detected but to a different level depending on diet composition.


The c-src tyrosine kinase (CSK) gene, a potential antioncogene, localizes to human chromosome region 15q23----q25.

We have previously reported the cloning of a novel cytoplasmic tyrosine kinase, CSK. This tyrosine kinase has been shown to downregulate the tyrosine kinase activity of the c-src oncoprotein through tyrosine phosphorylation of the c-src carboxyl terminus. Cell transformation by src oncoproteins is caused by several oncogenic mechanisms, which interfere with this phosphorylation. The CSK gene could therefore potentially function as an antioncogene. We have here mapped the CSK gene to 15q23----q25 by in situ hybridization.


Pharmacological agents affecting emesis. A review (Part II).

Part I of this article reviewed the pathophysiology of emesis, and its pharmacological treatment. Drug-induced vomiting was also discussed. In the second part of the review, other common causes of vomiting are considered. The basis of the use of antiemetics in morning sickness and migraine is still obscure; for the latter, serotonin 5-HT1 receptor agonists, 5-HT3 receptor antagonists and dopamine D2 receptor antagonists are effective. For motion sickness, control can be achieved with various antagonists of muscarinic or histamine H1-receptors. Centrally active adrenoceptor agonists in combination with a muscarinic antagonist or H1-receptor antagonist may offer better control of motion sickness and its associated symptoms than either antagonist alone; based on clinical studies, postoperative vomiting after opiate administration appears to be controlled by blocking dopamine D2, histamine H1- or muscarinic receptors. Radiation therapy appears to be similar to cytotoxic therapy in that the mediators produced or released by radiation activate both peripheral and central sites involved in the vomiting reflex. Blockade of dopamine D2 and 5-HT3 receptors may be effective.


Recent advances in pharmacological management of hypertension in diabetic patients with nephropathy. Effects of antihypertensive drugs on kidney function and insulin sensitivity.

Hypertension is often seen in Type 1 and Type 2 diabetic patients, particularly in those with nephropathy, and the progression of diabetic nephropathy is closely related to blood pressure elevation. Thus, the effects of antihypertensive drugs on kidney function and insulin sensitivity in diabetic patients are of great clinical importance. Successful antihypertensive treatment has been shown to slow the progression of diabetic nephropathy. Several results from short term studies have suggested that angiotensin converting enzyme (ACE) inhibitors may be advantageous over other conventional antihypertensive agents in reducing albuminuria in both hypertensive and normotensive diabetics with microalbuminuria or persistent proteinuria. However, the decline in glomerular filtration rate during ACE inhibitor treatment is comparable to that during effective treatment with conventional antihypertensive drugs in hypertensive Type 1 diabetic patients with overt nephropathy. Whether ACE inhibitors possess a specific effect in preventing the development of diabetic nephropathy remains to be seen in properly designed long term studies. Although calcium antagonists may preserve kidney function or possess a renoprotective effect in hypertensive Type 2 diabetics with nephropathy, firm evidence supporting this contention seems to be lacking and also requires long term evaluation. Increasing attention is being directed toward the effect of antihypertensive drugs on insulin sensitivity in diabetic patients: ACE inhibitors and alpha 1-adrenoceptor blocking agents have been shown to improve this sensitivity. Despite the widespread involvement of calcium in hormone secretion and action, calcium antagonists appear to have little effects on the glucoregulatory and calcium-regulatory hormones within the drug dosages used in clinical practice. Several clinical variables, such as the presence or absence of hypertension, overt nephropathy and microalbuminuria, or a combination of variables should be accounted for when evaluating critically the cumulative data on the effects of antihypertensive drugs on kidney function and albuminuria in the variety of diabetic patient groups. Understanding the pharmacokinetic and pharmacodynamic characteristics of antihypertensive drugs will be of clinical importance in diabetic patients with advanced nephropathy (glomerular filtration rate of less than 30 ml/min) and/or other complications, such as impaired gastric motility or gastroparesis, and will thereby lead to a more rational management of hypertension in those patients.


Guidelines for the treatment of vitiligo.

At present, there is no universally effective drug for vitiligo therapy; there are, however, various therapeutic modalities that may be beneficial. Therapeutic regimens used to treat vitiligo include psoralens and ultraviolet A light (PUVA), topical corticosteroids, fluorouracil locally applied with skin abrasion, a variety of surgical techniques to transplant autologous melanocytes from pigmented skin to nonpigmenting areas, a new photochemotherapeutic regimen using oral khellin with UVA phototherapy, and a recently proposed treatment with oral phenylalanine in combination with UVA exposure. PUVA and topical corticosteroids are the 2 most frequently used modalities. The use of effective sunscreens with a high sun protection factor (SPF) is of help in preventing the vitiliginous areas from burning and normal skin from becoming tanned. Cosmetic camouflage is also useful to disguise the white areas of skin. Finally, depigmentation should be considered in patients with greater than 50% cutaneous involvement who fail to respond or are unwilling to undergo treatment. The selection of patients for therapy should take into consideration the patient's motivation, the psychological impact of the disease and the clinical presentation of vitiligo, and should weigh the risks and benefits of prolonged therapy.


Cancer-associated anorexia and cachexia. Implications for drug therapy.

Anorexia and cachexia are major problems in patients with cancer. Such measures as anti-cancer therapy, dietary counselling or hyperalimentation are not very successful in reversing this phenomenon in the vast majority of cancer patients. Thus, several drugs have been evaluated as agents to ameliorate cancer-associated anorexia/cachexia. Cyproheptadine is an antiserotonergic drug which appears to cause slight appetite stimulation in patients. A randomised clinical trial, however, was unable to demonstrate any weight gain from this agent. Corticosteroids are frequently used in clinical practice for appetite stimulation in patients with advanced malignancies. Supporting this practice, 4 randomised clinical trials showed that corticosteroid medications can stimulate the appetites of advanced cancer patients. However, these studies were not able to show any substantial nonfluid weight gain in treated patients. Megestrol acetate is a progestational agent which appears to be a relatively potent appetite stimulant. Randomised studies in advanced cancer patients have shown both substantial appetite stimulation and improvement in the nonfluid bodyweights of patients receiving this drug. Preliminary evidence also suggests that this drug has antiemetic properties. Several clinical studies are currently ongoing to determine the effect of various doses of megestrol acetate in patients with cancer. Efforts are also ongoing to evaluate both anabolic steroids and hydrazine sulfate as drugs for the treatment of patients with cancer anorexia/cachexia. The preliminary nature of these investigations, however, precludes recommendations for the use of either of these latter 2 drugs in routine clinical practice.


The use of sedative agents in critically ill patients.

The main aim of sedation in the critically ill patient is to provide relief from anxiety and pain. The current, ideal level of sedation should leave a patient who is lightly asleep but easily roused. No single regimen is suitable for all patients. The level of sedation should be monitored, and the choice of agent, the dose and the route of administration adjusted appropriately. Midazolam is often used to provide sleep and anxiolysis. Alternatives include propofol and isoflurane. Propofol is easily titrated to achieve the desired level of sedation, and its effects rapidly end when the infusion is stopped. Isoflurane also appears promising, but special equipment is needed for its administration. Morphine is the standard analgesic agent. The principal metabolites, morphine-6-glucuronide, is also a potent opioid agonist and may accumulate in renal failure. Of the newer analgesic agents, alfentanil is an ideal agent for infusion, and may be the agent of choice in renal failure. Neuromuscular blocking agents are indicated only in specific circumstances, and used only once it is known patients are asleep and pain free. The actions of these agents are unpredictable in the critically ill patient. Alterations in drug effect and elimination may occur, especially in the patient with hepatic and renal failure. This may also apply to active metabolites of the parent drug. When planning sedation regimens, specific patient needs and staffing levels must be remembered. Attention to the environment is also important. Midazolam and morphine given by intermittent bolus or by infusion are the mainstay of most regimens. Propofol is ideal for short periods of care on the ICU, and during weaning when longer acting agents are being eliminated.


Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). A review of its pharmacological properties and prospective role in the management of myelosuppression.

Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) is a polypeptide hormone produced through recombinant DNA technologies in glycosylated (yeast or mammalian expression systems) or nonglycosylated (Escherichia coli expression system) form. It is a multilineage haematopoietin which stimulates proliferation and differentiation of bone marrow myeloid progenitors and increases peripheral white blood cell counts when administered systemically. Treatment is generally well tolerated, although mild to moderate flu-like symptoms are common and rGM-CSF-induced fever and fluid retention may be problematic in occasional patients. rGM-CSF accelerates recovery of peripheral neutrophil counts after bone marrow transplantation, and results of a placebo-controlled randomised trial correlate this with reduced infectious episodes and shortened length of hospitalisation in patients with lymphoid malignancies. A substantial number of patients with graft failure after bone marrow transplantation also respond to rGM-CSF. The duration of myelosuppression secondary to cancer chemotherapy can be significantly reduced by rGM-CSF which has permitted investigation of antineoplastic dose-intensity escalation. In some haematopoietic disorders (e.g. aplastic anaemia, myelodysplasia and neutropenia secondary to HIV infection and antiviral therapy), rGM-CSF produces clinically useful increases in peripheral blood granulocyte counts, although the effect is generally not sustained after drug withdrawal. The potential for rGM-CSF to stimulate proliferation of the abnormal clone in myelodysplasia and in acute myelogenous leukaemia following induction therapy is of concern. Available data suggest, however, that with appropriate monitoring and exclusion of high-risk patients this serious potential risk can be avoided, and that myelopoiesis is enhanced in such patients by rGM-CSF treatment. Recombinant colony-stimulating factors are a new therapeutic modality; hence many aspects of their use remain to be clarified. Nonetheless, as one of a small group of novel agents rGM-CSF has major potential in the management of myelosuppression secondary to cytoreductive therapy with or without bone marrow transplantation, and in amelioration of disturbed myelopoiesis. It represents an important application of biotechnology to a difficult area of therapeutics.


Moclobemide. A review of its pharmacological properties and therapeutic use in depressive illness.

Moclobemide is a reversible and selective inhibitor of the enzyme monoamine oxidase (MAO) subtype A with a broad spectrum of antidepressant activity. Controlled clinical studies suggest that the short term clinical efficacy of moclobemide is significantly superior to that of placebo, and comparable to that of the tricyclic antidepressants clomipramine, amitriptyline, imipramine and desipramine, the irreversible MAO inhibitor tranylcypromine and the second-generation antidepressants maprotiline, mianserin and fluvoxamine in the treatment of major depressive illness. Moclobemide appears to be equally effective in endogenous and nonendogenous depression, producing marked amelioration of clinical features of psychomotor retardation and depressed mood. Moclobemide is well tolerated, being largely devoid of the anticholinergic adverse effects, symptomatic postural hypotension and weight gain variously associated with the tricyclic antidepressants and irreversible MAO inhibitors, and appears considerably safer on overdosage than the tricyclic and second generation antidepressants. Moreover, moclobemide offers the advantage over the older, irreversible MAO inhibitors of causing only minimal potentiation of the pressor response to dietary tyramine (the so-called 'cheese effect'). Consequently, the risk of potentially fatal hypertensive crisis, a major deterrent to the wider acceptance of these earlier compounds, is substantially reduced with moclobemide, and the need for dietary precautions is minimised. With its efficacy against endogenous and nonendogenous depression, relatively rapid onset of antidepressant activity, and absence of carry-over effects on treatment withdrawal, moclobemide is likely to make an important contribution to the treatment of major depressive illness. Its favourable tolerability profile, safety on overdosage and beneficial effect on age-related cognitive impairment may be of particular value in the elderly and those with concurrent physical illness.


Acitretin. A review of its pharmacology and therapeutic use.

Acitretin (etretin), a second generation monoaromatic retinoid for use in the treatment of severe psoriasis and other dermatoses, is the major active metabolite of etretinate and possesses a similar therapeutic index; i.e. a similar ratio of clinical efficacy to adverse effects. When used alone at a maintenance dosage of 30 to 50mg daily, acitretin is effective in the treatment of psoriasis, causing a reduction in the severity of scaling, erythema and induration. Efficacy appears to be further enhanced by combination with psoralen-ultraviolet A photochemotherapy (PUVA) or ultraviolet B irradiation (UVB). These combinations reduce the time to lesion clearance and reduce the total radiation dose, improving overall safety. Comparative studies have confirmed the equivalence of acitretin and etrtinate with regard to efficacy and toxicity. Adverse reactions are dose-related and generally typical of hypervitaminosis A. Alopecia and mucocutaneous symptoms such as cheilitis and drying of the mucous membranes are particularly prevalent. Hypertriglyceridaemia and elevation of cholesterol levels also occur. Examination of the pharmacokinetic profile of acitretin reveals its main advantage over etretinate. Acitretin is less lipophilic than etretinate, and its lack of sequestration into 'deep' fatty storage sites is reflected in a comparatively short terminal elimination half-life of 50 to 60 hours, compared with 120 days for etretinate. Due to its teratogenic potential, acitretin is strictly contraindicated in women of childbearing potential unless effective contraceptive measures are employed. Etretinate has been identified in plasma samples of some patients treated with acitretin. Thus, acetretin has an established place in the treatment of keratinising disorders, although its use in women of child-bearing potential must be accompanied by effective contraceptive measures, with a further 2-year contraceptive period after therapy completion.


Prostaglandin E1 inhibits collagenase gene expression in rabbit synoviocytes and human fibroblasts.

Cartilage breakdown, as seen in inflammatory and degenerative joint diseases, can be mediated by proteolytic enzymes, such as the metalloproteinase collagenase, the only enzyme able to digest collagen at neutral pH. In vitro collagenase gene expression can be stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. We have investigated the effect of prostaglandin E1 (PGE1) on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated collagenase mRNA levels in the rabbit synoviocyte cell line HIG-82. PGE1, but not PGE2 or PGF2 alpha, was able to selectively reduce collagenase mRNA levels in a dose-dependent fashion. PGE1 markedly increased intracellular levels of cAMP, while PGE2 and PGF2 alpha had little or no effect on cAMP production in the HIG-82 synoviocytes. Agents known to increase intracellular cAMP levels, such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), mimicked the effect of PGE1, on collagenase mRNA levels. PGE1, forskolin, and IBMX also decreased collagenase mRNA levels in human skin fibroblasts, demonstrating that this observation was not unique to the HIG-82 cell line. Transient transfection experiments carried out in HIG-82 cells using a 1.2-kilobase portion of the 5'-flanking region of the human collagenase gene linked to the reporter gene luciferase demonstrated that PGE1, forskolin, and IBMX exert their inhibitory effect on the promoter region of the collagenase gene.


Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.

We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.


Mitogenic effect of a factor from rat somatomammotrophs on mammary epithelial cells.

We have previously established a somatomammotroph cell line (rPC0) derived from normal rat pituitary that secretes GH and PRL. In this study we report that conditioned medium from rPC0 cells (CM-rPC0) exhibited a stimulatory effect on the growth of rat mammary epithelial cells in culture. Fractionation of CM-rPC0 revealed that the growth-promoting activity of CM-rPC0 was associated with a fraction eluting from a diethylaminoethyl-Sephacel column at 0.5 M NaCl. The growth-promoting activity of this fraction was abolished by trypsin and was resistant to dithiothreitol treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the predominant components of the 0.5-M NaCl fraction were proteins migrating at the 14-18K region of the gel. The 0.5-M NaCl fraction did not contain immunodetectable GH or PRL. Further fractionation of CM-rPC0 on a heparin-agarose column showed that the growth-promoting activity eluted at 0.9 M NaCl. The two predominant proteins in this fraction had apparent mol wt of 14.5K and 18.2K. Based on the structural and biological properties, several known hormones and growth factors were excluded from consideration as potential candidates responsible for the growth-promoting effect of the 14-18K proteins. It is postulated that this group of 14-18K proteins contains a new factor(s) that affects the growth of mammary epithelial cells.


Expression of a lactogen-dependent insulin-like growth factor-binding protein in cultured mouse mammary epithelial cells.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.


Insulin-like growth factor binding protein (IGFBP)4 accounts for the connective tissue distribution of endothelial cell IGFBPs perfused through the isolated heart.

Insulin-like growth factor binding protein 4 (IGFBP4) was purified to homogeneity from conditioned media of bovine pulmonary artery endothelial cells and shown to have the N-terminal amino acid sequence DEAIHCPPCS, a sequence unique to IGFBP4. The IGFBP4 was separated into predominantly glycosylated and nonglycosylated fractions, with each fraction separately perfused through isolated, beating rat hearts. Both forms of IGFBP4 crossed the capillary boundary of the heart and distributed primarily in subendothelial connective tissue components with a connective tissue/cardiac muscle distribution ratio of 20:1 for the glycosylated fraction and 27:1 for the nonglycosylated fraction. Perfused IGFBP1, 2, 3, and IGF-I also crossed the capillary boundary but in contrast to IGFBP4, preferentially localized in cardiac muscle with a connective tissue/muscle ratio of approximately 1:3. We conclude that the connective tissue distribution previously reported for IGFBPs in conditioned media of pulmonary artery endothelial cells is due to IGFBP4.


Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.


The role of the oligosaccharide chains of thyrotropin alpha- and beta-subunits in hormone action.

TSH, a dimeric glycoprotein hormone, has two N-linked complex-type oligosaccharide chains on the alpha-subunit and one on the beta-subunit. The oligosaccharide chains of TSH are important for the expression of hormonal activity, but the contribution of those on each of the subunits to the activity is not clear. In the current study we have determined the relative importance of the oligosaccharide chains of TSH subunits using a recently reported method of enzymatic deglycosylation. The alpha- and beta-subunits of bovine TSH were deglycosylated with endoglycosidase-F and recombined to obtain differentially deglycosylated TSH. The derivatives showed no differences in their receptor-binding activities. The in vitro bioactivity of these derivatives was assessed by measuring adenylyl cyclase activity in bovine thyroid membranes and stimulation of cAMP production and growth in FRTL-5 cells. In the adenylyl cyclase assay, deglycosylation of the alpha-subunit alone had a more profound effect on the activity [maximal stimulatory activity (Vmax), 13% that of alpha.beta) than when the beta-subunit alone was deglycosylated (Vmax, 50% that of alpha.beta). In the FRTL-5 assays, removal of carbohydrate from TSH alpha, but not the beta-subunit, caused a 2- to 3-fold increase in the concentration required for half-maximal stimulation, with minimal change in the apparent Vmax. The adenylyl cyclase assay in bovine membranes was more sensitive to carbohydrate removal than the assays of rat FRTL-5 cells, in which the derivatives with lower activity were able to stimulate cAMP and growth to near-maximal levels, albeit at 3-fold higher concentrations. These results indicate that the carbohydrate chains in both subunits of TSH, particularly those in the alpha-subunit, are important in hormone action. In contrast to previous reports, our study shows that the beta-subunit plays a role in signal transduction.


Effect of epithelium removal and of enkephalin inhibition on the bronchoconstrictor response to three endothelins of the human isolated bronchus.

The three endothelins ET-1, ET-2 and ET-3 induced a potent contractile response in the human isolated bronchus with an intact epithelium, which proceeded on two different stages. The first stage was observed at low concentrations (high potency) (10(-12) to 10(-9) M) but corresponded to a low intrinsic activity (Emax maximal effect induced by ACh 10(-3) M), the second stage appeared at higher concentrations and corresponded to higher intrinsic activity. The rank order of potency for the two stages of contractile activity was ET-1 greater than ET-2 = ET-3. Removal of the epithelium significantly enhanced the two stages of the contractile responses to the three endothelins and abolished the differences in potency efficacy that were observed between ET-1, ET-2 and ET-3 when the epithelium was present. Phosphoramidon (10(-5) M), an enkephalinase inhibitor, was as potent as epithelium removal in enhancing the contractile responses to these agonists at low concentrations (first stage of contraction, 10(-16) to 10(-9) M). However, with high concentrations of endothelins (greater than 10(-9) M, second stage of contraction), phosphoramidon was less potent than epithelium removal in enhancing the contractile responses. In epithelium-denuded strips, preincubation with phosphoramidon did not further increase the maximal contractions induced by/or the potencies of ET-1, ET-2 or ET-3. After epithelium removal, responses to low doses of endothelins were attenuated by nicardipine (10(-6) M) whereas responses to high doses of the endothelins were not affected, as was also observed when the epithelium was present. (ABSTRACT TRUNCATED AT 250 WORDS)


Tachykinin-induced dyspnea in conscious guinea pigs.

Aerosol administration of neurokinin A (NKA) or substance P (SP) to conscious guinea pigs produced labored abdominal breathing (dyspnea). Time to onset of dyspnea was inversely related to tachykinin concentration. Aerosol administration of the neutral endopeptidase inhibitor thiorphan significantly potentiated tachykinin-induced dyspnea without affecting responses to leukotriene D4 (LTD4), carbachol, histamine, platelet activating factor or serotonin (5-HT), indicating selectivity for tachykinins rather than a nonspecific effect on agonist reactivity. The rank order of potency for producing dyspnea was LTD4 greater than or equal to NKA (with thiorphan) much greater than SP (with thiorphan) greater than 5-HT = carbachol greater than histamine greater than platelet-activating factor. Pretreatment with propranolol, phentolamine, methysergide, pyrilamine or the peptide leukotriene antagonist, ICI 198,165, did not alter dyspnea induced by NKA or SP. The dose-response curves for NKA and SP were shifted to small degrees (less than 3-fold) to the right by atropine and to the left by indomethacin. Also, pretreatment with capsaicin did not affect responses to NKA or SP, indicating that they do not cause dyspnea by activating capsaicin sensitive C-fibers. These results suggest primarily direct effects of NKA and SP. This model may be useful for in vivo evaluation of tachykinin antagonists.