VVA-labelled cells in monkey visual cortex are double-labelled by a polyclonal antibody to a cell surface epitope.

The staining patterns produced by the lectin Vicia villosa and by a commercially available polyclonal antibody generated to substance P were analysed and compared in monkey visual cortex at the light and electron microscopic levels. Vicia villosa lectin labels the cell surface of a subpopulation of cortical cells, producing a meshlike pattern over the soma and proximal dendrites. The polyclonal antibody labels three distinct elements in the cortex: a pericellular epitope present on a subpopulation of non-pyramidal cells, and putative intracellular sites in a type of small pyramidal cell located at the layer 5/6 border, and in a small number of non-pyramidal cells in the underlying white matter. Because of the similarity of the appearance of the Vicia villosa lectin labelling and the pericellular labelling produced by the polyclonal antibody, further experiments were conducted to determine the relationship between the cell surface sites recognized by these markers. Double-labelling experiments show that both sites are present on the same population of cells, and at the ultrastructural level both markers appear to outline the intersynaptic cell membrane, sometimes extending around presynaptic elements. However, preadsorption experiments indicate that the markers recognize different sites on the cell membrane. Preadsorption experiments also show that the pericellular epitope recognized by the polyclonal antibody is unlikely to be substance P, but it may be structurally similar to keyhole limpet haemocyanin. Comparison of cortical and subcortical staining patterns produced with the polyclonal antibody and with a commonly used monoclonal antibody to substance P reveal that one of the putative intracellular epitopes recognized by the polyclonal antibody is likely to be substance P.


Suramin: a novel growth factor antagonist with activity in hormone-refractory metastatic prostate cancer.

Suramin is known to inhibit the growth of malignant prostate carcinoma cells in vitro. This led us to evaluate the effectiveness of suramin in the treatment of 38 patients with prostate carcinoma refractory to hormone therapy. Suramin was administered by continuous infusion at a rate designed to reach a peak of 300 micrograms/mL at the end of 14 days. Patients were given 8 weeks to recover from any toxicity before beginning the second cycle. Subsequent cycles were administered in the same manner except the starting dose rate was 280 mg/m2. In 17 patients with measurable soft tissue disease, three had complete disappearance of soft tissue disease for 4, 5, and 11 months, whereas three patients had a greater than or equal to 50% decrease in the sum of the products of the diameters of all measurable disease for greater than or equal to 1 month. Of these 17 patients, pretreatment prostate-specific antigen (PSA) decreased by 75% or more in five (29%) and normalized in one (6%). The remaining 21 patients had disease limited to bone, and only one of these experienced resolution of more than 50% of all lesions on bone scan. Of these 21 patients, pretreatment PSA decreased by 75% or more in eight (38%) and normalized in five (25%). Median time to progression for all patients was 26.3 weeks, and median survival was 42.3 weeks. Patients with bone involvement alone exhibited a better survival than patients with soft tissue involvement (P2 = .02). Survival was strongly correlated (P2 = .0001) with a decline in the pretreatment PSA of greater than or equal to 75% by the eighth week on therapy, with nearly an 85% survival at 1 year compared with a 20% survival for those whose pretreatment PSA did not decline by that amount. We conclude that suramin is an active agent in hormone-refractory prostate carcinoma.


Cisplatin-based combination chemotherapy in the treatment of poorly differentiated carcinoma and poorly differentiated adenocarcinoma of unknown primary site: results of a 12-year experience.

We previously reported excellent responses to cisplatin-based chemotherapy in a minority of patients with poorly differentiated carcinoma (PDC) or poorly differentiated adenocarcinoma (PDA) of unknown primary site. We have continued to study and to treat these patients, and now report clinical characteristics, treatment results, and prognostic factors in a large group of patients identified prospectively. Between February 1978 and December 1989, we treated 220 patients with PDC or PDA of unknown primary site. The median age was 39 years; 48% of patients had predominant tumor location in the mediastinum, retroperitoneum, or peripheral lymph nodes. Specialized pathologic studies resulted in the identification of specific tumor types in only a few cases. All patients received cisplatin-based chemotherapy; between 1978 and 1984, 116 patients received cisplatin, vinblastine, and bleomycin (PVeB) +/- doxorubicin, and 104 patients treated since January 1985 received cisplatin and etoposide +/- bleomycin. One hundred thirty-eight patients (63%) had objective responses to therapy, and 58 (26%) had complete response. Thirty-six patients (16%) are currently disease-free at a median of 61 months following therapy (range, 11 to 142 months). Actuarial 10-year survival is 16%. Favorable prognostic factors identified by Cox regression analysis include: (1) predominant tumor location in the retroperitoneum or peripheral lymph nodes, (2) tumor limited to one or two metastatic sites, (3) no history of cigarette use, and (4) younger age. Patients with PDC or PDA of unknown primary site represent another group of patients for whom potentially curative therapy is available. Patients with this syndrome should be distinguished from patients with well-differentiated adenocarcinoma of unknown primary site, and should receive a trial of cisplatin-based chemotherapy.


Vasoactive intestinal polypeptide modulates GABAA receptor function in bipolar cells and ganglion cells of the rat retina.

1. The effect of vasoactive intestinal polypeptide (VIP) on bipolar cells and ganglion cells freshly dissociated from the rat retina was studied under voltage clamp with the use of patch-clamp recording in the whole-cell configuration. 2. Application of VIP (1-100 microM) by itself resulted in no detectable current response in either bipolar cells or ganglion cells. However, gamma-aminobutyric acid (GABA)-activated macroscopic current responses elicited in both neuronal populations were potentiated on superimposed exposure to the neuropeptide. 3. GABA-activated chloride currents and muscimol-induced current responses were similarly potentiated on exposure to VIP, suggesting a synergistic interaction between VIP and GABAA receptor mechanisms. 4. We postulate that VIP plays a neuromodulatory role by regulating the excitability of inner retinal neurons and in this way modulates the efficacy of synaptic transmission in the retina.


Porcine colostrum and milk stimulate visceral organ and skeletal muscle protein synthesis in neonatal piglets.

Our objective was to determine the relative contributions of protein synthesis and protein absorption in the rapid accretion of gastrointestinal protein in suckling piglets during the early neonatal period. We measured the rates of tissue protein synthesis using a flooding dose of L-[4-3H]phenylalanine in various visceral and peripheral tissues of neonatal piglets fed water, mature milk or colostrum for 6 h. The jejunal and ileal protein synthesis rates in piglets fed either colostrum or milk were three- to fourfold higher than in piglets fed water. The increased jejunal and ileal protein synthesis could not, however, account for the differences in protein mass between the colostrum-fed and water-fed groups. The relative abundance of IgG, a major porcine colostral protein, in jejunal tissue was markedly higher in piglets fed colostrum than in piglets fed either milk or water. The fractional protein synthesis rates in liver, kidney, spleen and skeletal muscle and the absolute protein synthesis rates in liver and spleen were also greater in piglets fed colostrum than in those fed milk or water. Increased endogenous protein synthesis made only a minor contribution to the increased intestinal protein accretion in neonatal piglets fed colostrum. A much larger proportion of this increase seemed to be a result of absorption and retention of ingested immunoglobulins.


The magnitude of the acute phase protein response is attenuated by protein deficiency in rats.

We assessed the growth rate and changes in plasma albumin, total protein and alpha 2-macroglobulin concentrations (a major acute phase protein in rats) before and after a subcutaneous injection of turpentine (0.5 mg/kg body wt) in groups of rats receiving one of a series of protein-deficient diets (protein concentrations of 0.5, 1.5, 3.0, 4.5 or 6.0 g/100 g) or a diet containing an adequate level of protein (20 g/100 g) for maximal growth. Increasing protein deficiency in the different groups of animals reduced the basal albumin and total protein concentrations and attenuated the total protein and alpha 2-macroglobulin responses to turpentine. Increasing protein deficiency delayed the time taken for alpha 2-macroglobulin to reach peak concentrations post-injection and its return to basal concentrations. The turpentine-induced hypoalbuminemia was similar in all groups of animals (approximately 10 g/L depression) but restoration to values that were present before turpentine injection was increasingly delayed with increasing protein deficiency. The magnitude of the acute phase response (peak alpha 2-macroglobulin concentration) was found to be directly related to growth rate (r = 0.70, P less than 0.001). We concluded that protein deficiency can alter the pattern and magnitude of the acute phase responses in circulating protein concentrations to an extent that is dependent on the severity of protein deficiency.


B-cell epitope mapping of the entire SIVagm envelope glycoprotein including fine mapping of immunogenic regions.

To allow the precise definition of the anti-lentiviral immune response in the natural host and to facilitate the development of an alternative animal model for vaccine development, we are identifying the immunogenic domains of SIVagm proteins. First, a total of 173 synthetic 15-mer peptides with an overlap of 10 amino acids were produced spanning the entire envelope glycoprotein of the molecular clone SIVagm3. These peptides were used as antigen in enzyme-linked immunosorbent assays for identifying regions recognized by antibodies from naturally infected African green monkeys and monkeys infected with a molecular clone. Regions corresponding to the HIV-1 V3, the transmembrane protein (TMP) "Gnann peptide", and the C-terminal area of the outer envelope protein were shown to be immunodominant. These regions were re-synthesized as 15-mer peptides with an overlap of 14 amino acids and used to precisely map the epitopes recognized. Sera from 93 captive and 61 wild animals were tested by SIVagm-specific Western blot (WB) and for ELISA reactivity against the immunodominant TMP peptide. One hundred percent (76/76) of the WB-positive captive animals and 98% (41/42) wild WB-positive animals also reacted against the peptide. In contrast, only 62% of the WB-positive sera reacted with the "V3" epitope and 46% with the gp130 C-terminal epitope.


CD20 expression is increased on B lymphocytes from HIV-infected individuals.

In studies presented here, we show that expression of the pan B cell marker CD20 is markedly increased on B lymphocytes from HIV-infected individuals and that this increase tends to be greater in individuals with more advanced disease. By using multiparameter FACS analyses to quantitate surface density of CD20 and intracellular glutathione (GSH) levels simultaneously, we further show that the distribution of intracellular glutathione (GSH) levels in B cells of HIV-infected individuals is more heterogeneous than in uninfected controls. Finally, we show that the intracellular GSH levels correlate with CD20 expression on a per-cell basis in all infected individuals. These findings suggest that CD20 expression, which can be precisely measured, may prove to be a useful surrogate marker for monitoring HIV infection.


Urticaria: new molecular insights and treatments. The Parkes Weber Lecture 1991.

Chronic urticaria remains one of the major unsolved clinical problems in dermatology. My group has employed an integrated experimental approach in order to shed light on the pathophysiology and treatment of this group of disorders. Using delayed pressure urticaria as a model, evidence has emerged of the role of eosinophil major basic protein (MBP) and of interleukin-6 (IL-6) as important molecular mediators, possibly explaining the poor response to H1 antihistamines. Our recent work in chronic idiopathic urticaria has led to identification of a circulating greater than 100 kD factor which causes wealing following intradermal injection and which releases histamine from normal leukocytes in vitro. Further characterisation confirmed that this skin and histamine releasing reactivity is due mainly to an IgG anti-IgE autoantibody. That this autoantibody is functionally significant is supported not only by its ability to release histamine and cause local wealing, but also by the results of removal by plasmapheresis which we have shown to cause clinical improvement in seven out of eight patients with severe unremitting chronic urticaria. It is concluded that chronic 'idiopathic' urticaria is an autoimmune disease due, in most patients, to a functionally significant IgG anti-IgE autoantibody. Immunotherapy offers the best long-term prospects of relief in severe unremitting cases.


Novel non-nucleoside inhibitors of HIV-1 reverse transcriptase. 2. Tricyclic pyridobenzoxazepinones and dibenzoxazepinones.

Dibenz[b,f][1,4]oxazepin-11(10H)-ones (III), pyrido[2,3-b][1,4]benzoxazepin-6(5H)-ones (IV), and pyrido[2,3-b]- [1,5]benzoxazepin-5(6H)-ones (V) were found to inhibit human immunodeficiency virus type 1 reverse transcriptase with IC50 values as low as 19 nM. A-ring substitution has a profound effect on activity, with appropriate substituents at the positions ortho and para to the lactam nitrogen providing dramatically enhanced potency. Substitution in the C-ring is generally neutral or detrimental to activity. Although a C-ring amino substituent at the position meta to the lactam carbonyl is generally beneficial to activity, it has essentially no effect when the A-ring is optimally substituted. Like the dipyridodiazepinone nevirapine, compounds III-V are specific for HIV-1 RT, exhibiting no inhibitory activity against HIV-2 RT or other virial reverse transcriptase enzymes.


Modulation of transcription in rat liver nuclei in vitro by a diol epoxide of benzo[a]pyrene.

Treatment of isolated rat liver nuclei with 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene, the ultimate carcinogenic metabolite of benzo[a]pyrene, resulted in inhibition of transcription as measured by radioactive precursor incorporation into RNA. The mechanism of inhibition as analyzed by use of different types of inhibitors suggested that the carcinogen acted on both the major components of transcription machinery, that is, the template chromatin and the enzyme RNA polymerases. This action correlates well with the observations made after administration of benzo[a]pyrene to rats.


Renal excretion of perfluorooctanoic acid in male rats: inhibitory effect of testosterone.

There is a marked sex difference in the whole-body elimination of perfluorooctanoic acid (PFOA) in rats, with females excreting the perfluorinated acid much more rapidly (half life [t1/2] less than 1 day) than males (t1/2 = 15 days). Our objective was to determine if androgens or estrogens are involved in causing this sex difference in PFOA elimination. Castration of males greatly increased the elimination of [1-14C]PFOA (9.4 mumol/kg, i.p.) in urine, demonstrating that a factor produced by the testis was responsible for the slow elimination of PFOA in male rats. Castration plus 17 beta-estradiol had no further effect on PFOA elimination whereas castration plus testosterone replacement at the physiologic level reduced PFOA elimination to the same level as rats with intact testes. Thus, in male rats, testosterone exerts an inhibitory effect on renal excretion of PFOA. In female rats, neither ovariectomy nor ovariectomy plus testosterone affected the PFOA urinary elimination, demonstrating that the inhibitory effect of testosterone on PFOA renal excretion is a male-specific response. Probenecid decreased the high rate of PFOA renal excretion in castrated males but had no effect on male rats with intact testes. We conclude that testosterone is a key determinant of the sex difference in PFOA elimination in rats.


Fine structure of the dorsal lingual epithelium of the little tern, Sterna albifrons Pallas (Aves, Lari).

The dorsal surface of the tongue of the little tern, Sterna albifrons, has a distinctive anterior region for five-sixths of its length and a terminal posterior region. The anterior region observed by scanning electron microscopy is distinguished along its forward half by a median line from which median papillae protrude. The hind half of the anterior region has a median sulcus without papillae. The deciduous epithelium on both sides of the median line and sulcus bears scattered epithelial protrusions. The posterior lingual region has neither median papillae nor deciduous epithelium. So-called giant conical papillae are located in a transverse row between anterior and posterior regions. Delicate microridges adorn the surfaces of all outer epithelial cells in both regions. Examination of the dorsal lingual epithelium by light and electron microscopy provides histologic and cytologic criteria for distinguishing anterior and posterior regions. Basal cells are nearly alike throughout the dorsal epithelium. Intermediate layer cells of the anterior region contain numerous tonofibrils in electron-dense bundles composed of 10 nm tonofilaments. The outer layer is composed of electron-dense, well-keratinized cells, and electron-lucent epithelial protrusions are present on the exposed surface of the outermost cells. Median papillae are composed of typical keratinized cells, which are nearly filled with keratin filaments. Intermediate layer cells in the posterior region of the tongue are nearly filled with unbundled tonofilaments. There is only a very thin outer keratinized layer in this region.


Teratogenic and macromolecular synthesis inhibitory effects of trimethylamine on mouse embryos in culture.

Trimethylamine (TMA) is an aliphatic amine, and its blood levels can increase after ingestion of certain foods, such as fish, and during disease states, such as chronic renal failure. We recently reported that TMA can inhibit fetal development in vivo and in vitro in mice. The present studies were done to find out if the inhibitory effects of TMA on embryonic development are caused by a decrease in macromolecular synthesis, using mouse embryo cultures as the experimental model. At a submaximally toxic concentration (0.75mM), TMA inhibited the growth of embryos to approximately 70% of control and caused neural-tube defects in 73% of embryos. By 42 h of culture, DNA, RNA, and protein content of TMA-treated embryos were approximately 50% of the control values. Embryotoxic effects of TMA were not caused by changes in pH and osmolarity of the culture media. The inhibitory effects of TMA on embryonic growth were time dependent and apparent at 2-4 h of culture. The inhibition of growth was accompanied by a decrease in the incorporation of tritium-labeled thymidine, uridine, and leucine into DNA, RNA, and proteins, respectively. Thiols (L- and D-cysteine, glutathione) and the antioxidant L-ascorbic acid did not cause significant antagonism of embryotoxic effects of TMA. It is concluded that TMA exerts teratogenic effects on mouse embryos in culture and inhibits their growth by reducing macromolecular synthesis; these effects may not involve glutathione depletion or generation of free radicals.


Partially hepatectomized rats: a model for the study of the effect of toxins on the plasma protein profiles of nascent hepatocytes.

A useful framework is proposed for unifying the synthesis of plasma proteins and their degradation by, or release from, liver cells of intact and partially hepatectomized rats, in which synthesis and release of acute-phase plasma proteins occur in synchrony with the internalization and catabolism of plasma and extracellular proteins. The catabolism of proteins and other hepato-intracellular glycoproteins during sepsis or trauma is essential to provide constituent amino acids and carbohydrates for the synthesis of acute-phase plasma proteins. Increases in the plasma levels of acute-phase response proteins in sham-operated rats reached a maximum between 1 and 2 d after mock surgery, and had returned virtually to control levels within 6 d. By contrast, acute-phase proteins in the plasma of partially hepatectomized rats were decreased by 10-20% of their initial values after 24 h. A maximum acute-phase response on d 7 after the operation was characterized by an increase of 181, 445, and 19% for alpha-1-acid glycoprotein, hepatoglobin, and hemopexin, whereas other acute-phase proteins remained below control levels, for example, by 11, 25, and 38% for albumin, transferrin, and prealbumin, respectively. This delayed response suggests that the nascent liver cells had inherited the capacity of the parent cells to respond to inflammatory signal and had synthesized acute-phase plasma proteins. Accordingly, a time frame for the application of toxin to nascent hepatocytes is suggested. An increased activity (300 +/- 50%) for both bound and free neuraminidase in remnant liver tissue 19 h post partial hepatectomy suggested that hepatic regenerating factor(s) were produced in liver tissue via the hepatic bound and/or free neuraminidase-mediated desialylation of humoral substrates. By contrast, circulating levels of lysosomal enzymes alpha-fucosidase and beta-N-acetyl-D-glucosaminidase were increased marginally after 24 h but had returned nearly to control levels after 7 d, suggesting that lysosomal acid hydrolases do not play a major role in regenerative DNA synthesis, mitosis, or in the synthesis of acute-phase plasma proteins.


[Histopathological characteristics of rheumatoid arthritis--as a clue to elucidate its pathogenesis].

A correct histopathological diagnosis of Rheumatoid Arthritis (RA) is quite important for the decision of early phase treatment to cure it fundamentally. But, generally speaking, usual hospital pathologist is not so much experienced about RA. The purpose of this article is originally to let such pathologist familiar in RA pathology, but for the RA specialist to offer any clue to elucidate the still-unknown etio-pathogenesis of RA or to cure RA fundamentally. The "Tetralogy of RA Arthritis for pathologist" must be as follows: (1) Enormous proliferation of well-permeable granulation-tissue-type neo-vascularization, some of which became high column-endothelial and the center of primary as well as secondary follicle-like lymphoid cell cluster. (2) Lymphoid cluster in RA synovium is also pathological in function. It consisted of preferentially CD4T and B cells to produce IgG rheumatoid factor endlessly. (3) Synovial lining A and B cells proliferate as far as five layers of each, but later, the sublining D (M) and D (F) cells proliferate more and more and finally replace the lining cells. D (M) cells express macrophage marker and full of lysosome, on the contrary, D (F) cells express mesenchymal marker and contains much metalloproteinase. Both express strong Class II antigens but neither has complement activation inhibitor DAF. (4) Proliferation of these D cells with full of mesenchymal tissue destroying and inflammation accelerating activity must be playing a major role in the joint destruction of RA, some in shape of pannus and more in shape of granulation tissue in and around the bone.


[Effects of FK506 on aminonucleoside-induced nephrotic rats].

We examined the effect of immunosuppressive agent, FK506 (Fujisawa, Co.), on puromycin aminonucleoside (PAN)-induced nephrosis. Single i.p. injection of PAN in a dose of 100 mg/kg was introduced into Munich-Wistar rats weighing about 200 g. Those rats were divided into four groups. PAN-induced nephrosis rats in group 1 (PAN-FK0.1, n = 5), group 2 (PAN-FK0.3, n = 5), group 3 (PAN-FK1.0, n = 5) were treated with i.m. injection of FK506 for 10 days in a dose of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, respectively, and rats in group 4 were treated with FK-placebo (PAN-PL, n = 5). The rats in group 5 with Saline+placebo were served as a control (NS-PL, n = 5). Among 5 groups, urinary protein, anionic sites (AS) in GBM, and subsets of peripheral lymphocytes through FACS were compared. After 9 days of PAN injection, the rats in PAN-FK0.1 (160.0 +/- 38.4), PAN-FK0.3 (118.0 +/- 34.4) & PAN-FK1.0 (89.2 +/- 40.0) given FK-506 had significantly less proteinuria in a PAN dose dependent manner, compared to those in NS-PL (349 +/- 86.8 mg/day). The numbers of AS/1000 mmGBM were more attenuated in FK506-treated PAN rats (PAN-FK1.0; 16.2 +/- 3.9) than those in PAN-PL (11.7 +/- 4.4). In related to subset of lymphocytes, increased W3/25 in PAN-PL was regressed in PAN-FK0.1, PAN-FK0.3 & PAN-1.0 after 10 days of PAN-injection. W3/25/OX-8 was significantly higher in PAN-PL (3.6) than those in NS-PL (2.4), but not between PAN-1.0 & NS-PL. These data indicate that the mechanism for therapeutic effect of FK506 on PAN-induced nephrosis includes a revision of abnormal cellular immunity, which attenuates the decrease of AS structure and as a result decrease proteinuria.


Flow cytometric detection of rare normal human marrow cells with immunophenotypes characteristic of acute lymphoblastic leukemia cells.

Rare subpopulations of normal marrow B lymphoid cells expressing immunophenotypes typically found in B-lineage acute lymphoblastic leukaemias (ALL) were sought by multiparameter flow cytometry. First, CD34+ marrow leukocytes were isolated by immune adherence using immunomagnetic microspheres, and analyzed for coexpression of the following pairs of membrane antigens: CD34 CD22; CD34 CD20; and CD10 CD22. Terminal deoxynucleotidyl transferase expression was not assessed. All three antigen combinations were found on small percentages of the CD34-enriched cell population. Second, unseparated normal low density marrow leukocytes were examined by 'gating' on cells with the right-angle light scatter of lymphoid cells, plus either CD34+ or CD10+ immunofluorescence. This independent approach confirmed that rare subsets of normal cells coexpress 'immature' and 'mature' differentiation antigens. In addition, remission marrow cells were examined from two children who had completed therapy for ALL two and four months earlier. Both specimens had a more than threefold increase in CD34+ cells over normal marrow, and cells coexpressing immature and mature cell surface antigens were easily detected. These findings demonstrate that immunophenotypes characteristic of B-lineage ALL, previously labeled 'asynchronous' with respect to the developmental sequence of the majority of normal B lymphoid cells, exist at low frequency in normal human bone marrow.


Expression of surface adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) in adult acute leukemia: relationship with initial characteristics and prognosis.

Adhesion molecules CD58 and CD54 are involved in cell-cell interactions that are potentially important in the biology of acute leukemia (AL). Expression of these molecules was studied in 79 cases of adult AL including 50 cases of acute non-lymphoid leukemia (ANLL) and 29 cases of acute lymphoid leukemia (ALL) using an indirect immunofluorescence technique. CD58 was expressed in 45 +/- 26% of ANLL cells and 43 +/- 32% of ALL cells, and its expression did not correlate with any other marker. In ALL, the expression of CD58 was inversely correlated with the presence of a clinical tumoral syndrome (p = 0.0009), leucocytosis (p = 0.005), and the percent of peripheral blast cells (p = 0.001). The major finding in this study was the association between CD58 expression and prognosis. In ANLL, higher expression of CD58 was independently associated with higher CR rate (p = 0.04), longer overall survival (p = 0.02), and longer disease-free survival (p = 0.007). In ALL, higher expression of CD58 was associated with longer survival (p = 0.05). CD54 was expressed only on 17 +/- 16% of ANLL cells and 11 +/- 11% of ALL cells; its expression on ANLL was positively correlated with that of CD11 (p = 0.03), CD15 (p = 0.001) and CD34 (p = 0.01). CD54 expression did not correlate with clinical and hematologic characteristics. We conclude that the expression of adhesion molecule CD58, but not CD54, in AL is related to initial characteristics and evolution of the disease.


CD5+ B cells from bovine leukemia virus infected cows are activated cycling cells responsive to interleukin 2.

Most of the B cells from bovine leukemia virus (BLV) infected cows in persistent lymphocytosis (PL) were known to express the CD5 T-cell marker but it was not known whether this peculiar membrane phenotype relates to an activation state. It was demonstrated that these B cells were also flagged by two other membrane markers normally borne by cells belonging to the myeloid lineage (namely CD11b and CD11c). Moreover, cell cycle analysis illustrated that a significant percentage of these B cells (greater than 15%) left their resting (G0/G1) status and progressed through the cell cycle. In addition, T-cell-depleted peripheral blood mononuclear cells from animals in PL were shown to proliferate in response to a IL-2-containing supernatant (MLA 144). These results indicate that the CD5+ B cells from BLV-infected cows in PL are activated cells.


Recombinant human TNF-alpha stimulates the secretion of granulocyte colony-stimulating factor in vivo.

Tumor necrosis factor (TNF) is a macrophage-derived cytokine that causes hemorrhagic necrosis of several human tumors in vitro. It has a wide range of biologic effects including stimulation of secretion of both granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) by normal adult lung fibroblasts in culture. No in vivo data are available on the effect of exogenously administered TNF on cytokine production. In the studies reported here, we show that G-CSF accumulates in the serum in vivo in response to recombinant TNF (rTNF) administration. At the peak of the response circulating levels of 2-6 ng/ml of biologically active G-CSF are detectable. Surprisingly, circulating levels of GM-CSF, interleukin-3 as well as a number of other cytokines were not detectable within the limits of the assays. The results indicate that the levels of GM-CSF or interleukin-3 are minimally 100-fold lower than the peak levels of G-CSF. These data illustrate the complex interplay that cytokines have in vivo. Understanding these interactions in humans is crucial to the correct use of this new class of agents in the clinic.


Sensitivity and specificity of five different mycoplasma detection assays.

The sensitivity and specificity of five different mycoplasma detection tests were evaluated in comparison with the classical microbiological culture assay on agar plates as the reference method: direct fluorochrome DNA staining (direct DAPI), DNA staining of an indicator cell line (indirect DAPI), RNA hybridization with a cDNA specific for ribosomal mycoplasmal RNA, an enzyme-linked immunosorbent assay (ELISA) with mycoplasma-specific antibodies, and a biochemical cytotoxicity assay (6-MPDR). A large panel of continuous cell lines (20 adherent and 233 suspension cell lines, most of the latter were human leukemia-lymphoma cell lines) were analyzed for infection with mycoplasma. The results of the comparative analysis for sensitivity and specificity of the various tests were as follows: 100% and 100% for the indirect DAPI, 100% and 98% for the RNA hybridization assay, 87% and 94% for the direct DAPI, 72% and 100% for the ELISA, 75% and 90% for the biochemical 6-MPDR assay. Each of these approaches has both advantages and disadvantages with regard to cost, time, reliability, specificity, and sensitivity. The best compromise for routine mycoplasma testing is a combination of several techniques (e.g. direct culture on agar, RNA hybridization, and direct or indirect DAPI).


Effect of fluoxetine on serotonin and dopamine concentration in microdialysis fluid from rat striatum.

Fluoxetine injected i.p. into rats at a dose of 10 mg/kg rapidly increased serotonin concentration in microdialysis fluid from the striatum by at least 4-fold, an increase that was maintained throughout the 3 hr observation period. Dopamine concentration in the microdialysis fluid did not change. The concentration of the two dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was not changed in the microdialysis fluid, whereas the concentration of the serotonin metabolite, 5-hydroxyindoleacetic acid, was significantly decreased after fluoxetine injection. The increased extracellular concentration of serotonin no doubt resulted from inhibition of the serotonin uptake carrier by fluoxetine, and the lack of change in dopamine is evidence for the specificity of action of this uptake inhibitor.


Osmotic complications of low-molecular-weight dextran therapy in free flap surgery.

Low-molecular-weight dextran is utilized by many microsurgeons after free tissue transfer for its beneficial effects on the microcirculation. While it is generally felt to have a low rate of complications, severe complications secondary to dextran's osmotic effects may occur. We report here on two cases in which therapy with low-molecular-weight dextran was strongly implicated as a causative factor in major complications. These cases and their background are reviewed. Guidelines for the safe administration of this agent in patients post free-tissue transfer are discussed.


Histochemical discrimination of fibers in regenerating rat infraorbital nerve.

In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.


The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.


Translation initiation in Escherichia coli: sequences within the ribosome-binding site.

The translational roles of the Shine-Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied. The Shine-Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence. Each Shine-Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long. Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG. Translation was also affected by residues 3' to the initiation codon. The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon. The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation.


The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats.

Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C. fimi endoglucanases A and B.


Effect of galanin on growth hormone-releasing hormone-stimulated growth hormone secretion in adult patients with nonendocrine diseases on long-term daily glucocorticoid treatment.

Glucocorticoids are thought to inhibit growth hormone (GH) secretion through an enhancement of endogenous somatostatin tone. The aim of our study was to evaluate the effect of galanin, a neuropeptide that stimulates GH secretion, on GH-releasing hormone (GHRH)-induced GH secretion in adult patients with nonendocrine diseases who were under daily immunosuppressive glucocorticoid therapy. Six normal subjects (four men, two women) and seven steroid-treated subjects (three men, four women) were studied. GHRH-induced GH secretion was evaluated during a 40-minute intravenous (i.v.) infusion of saline or porcine galanin (12.5 micrograms/min). During saline infusion, steroid-treated patients showed a blunted GH response to GHRH (GH peak, 8.1 +/- 2.8 micrograms/L), as compared with normal subjects (GH peak, 23.8 +/- 3.9 micrograms/L). During galanin infusion, the GH response to GHRH was significantly enhanced (GH peak, 46.6 +/- 9.4 micrograms/L, P less than .05), as compared with saline infusion in normal subjects. In contrast, galanin infusion did not enhance the GH response to GHRH (GH peak, 16.6 +/- 6.5 micrograms/L), as compared with saline infusion in steroid-treated patients. The area under the GH-response curves was also significantly (P less than .05) lower in steroid-treated subjects, as compared with normal subjects. Thus, galanin failed to normalize or enhance the GH response to GHRH in patients treated long-term with glucocorticoids. It can be hypothesized that galanin does not elicit GH secretion by decreasing hypothalamic somatostatin tone.


Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells.

CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.


Regulation and interaction of multiple protein factors with the proximal promoter regions of a rice high pI alpha-amylase gene.

The alpha-amylase gene is known to be regulated by the plant hormone gibberellin (GA) in cereal aleurone cells. The accumulation of the mRNA corresponding to a rice high pI alpha-amylase gene, OSamy-c, was stimulated 20-fold by exogenous GA3 in half-seeds lacking embryos. Regulatory regions in the promoter of this high pI sub-family were analyzed. The OSamy-c 5' flanking sequence, spanning positions -231 to +29, was fused upstream of the beta-glucuronidase (GUS) gene coding region. The delivery of this plasmid into rice aleurone cells by the biolistic method resulted in a GA-stimulated synthesis of GUS. Gel retardation assays were performed to study protein-DNA interactions between putative regulatory sequences of OSamy-c and partially purified rice seed extracts. We identified multiple seed-specific protein factors that bind to proximal regions of the OSamy-c promoter between positions -231 and -162. Five different proteins were distinguished based on competitive binding studies. Three protein binding regions were located by footprinting analyses, one of which is located in the conserved sequence also found upstream of other GA-inducible genes. Two protein factors in rice aleurone cells that interact with the putative regulatory sequence do not require GA induction.


Multiple states of rat brain (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors as revealed by quantitative autoradiography.

The binding of (RS)-alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for non-N-methyl-D-aspartate excitatory amino acid receptors, was investigated in rat brain using an autoradiographic receptor binding technique. [3H]AMPA binding sites were widely distributed throughout the rat central nervous system, and the rank order of potency of displacers of [3H]AMPA binding was quisqualate greater than AMPA greater than 6,7-dinitroquinoxaline-2,3-dione = 6-cyano-7-nitroquinoxaline-2,3-dione greater than beta-N-oxalylamino-L-alanine greater than glutamate greater than kainate. Potassium thiocyanate (0-100 mM) exerted a 4-fold stimulation of [3H]AMPA binding, without changing the relative regional distribution of [3H]AMPA binding densities among rat brain regions. Scatchard analysis of equilibrium saturation binding revealed high affinity and low affinity components of [3H]AMPA binding, even in the absence of potassium thiocyanate. Addition of potassium thiocyanate increased the number of high affinity [3H]AMPA binding sites without a change in affinity. In addition, the number of low affinity [3H]AMPA binding sites was unchanged in the presence of potassium thiocyanate, but the affinity of low affinity [3H]AMPA binding was greatly increased. [3H]AMPA thus binds specifically to two affinity conformations of postsynaptic binding sites that appear to be interconverted with potassium thiocyanate. The pharmacologic profile of these sites is consistent with that of the ion channel-linked ("ionotropic") quisqualate/AMPA class of excitatory amino acid receptor in the rat central nervous system.


Stimulation of adenylate cyclase by amylin in CHO-K1 cells.

The CHO-K1 cell line responds to the peptide amylin by a rapid elevation of cAMP. The related peptide calcitonin gene-related peptide (CGRP) is 100 times less potent at stimulating adenylate cyclase than is amylin. The actions of amylin at this receptor are concentration dependent and not antagonized by the CGRP antagonist CGRP-(8-37). Although these cells have receptors for calcitonin, amylin is unable to take part in any high affinity interaction with these receptors, as assessed by radioligand binding. The CHO-K1 cell line has receptors for amylin that are distinct from those for calcitonin and CGRP.


Kynurenic acid analogues with improved affinity and selectivity for the glycine site on the N-methyl-D-aspartate receptor from rat brain.

The glycine site on the N-methyl-D-aspartate (NMDA) subtype of receptors for the excitatory neurotransmitter glutamate is a potential target for the development of neuroprotective drugs. We report here two chemical series of glycine site antagonists derived from kynurenic acid (KYNA), with greatly improved potency and selectivity. Disubstitution with chlorine or bromine in the 5- and 7-positions of KYNA increased affinity for [3H]glycine binding sites in rat cortex/hippocampus P2 membranes, with a parallel increase of potency for antagonism of NMDA-evoked responses in the rat cortical wedge preparation. The optimal compound was 5-I,7-Cl-KYNA, with an IC50 for [3H]glycine binding of 29 nM and an apparent Kb in the cortical wedge preparation of 0.41 microM. Reduction of the right-hand ring of 5,7-diCl-KYNA reduced affinity by 10-fold, but this was restored by substitution in the 4-position with the trans-phenylamide and further improved in the trans-benzylamide. The optimal compound was the transphenylurea (L-689,560), with an IC50 of 7.4 nM and an apparent Kb of 0.13 microM. Both series of compounds displayed a high degree of selectivity for the glycine site, having IC50 values of greater than 10 microM versus radioligand binding to the glutamate recognition sites of NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors and the strychnine-sensitive glycine receptor. Selectivity versus AMPA receptor-mediated responses was also apparent in the rat cortical wedge and in patch-clamp recordings of cortical neurons in culture. Experiments using [3H]dizocilpine (MK-801) binding indicated that 5,7-diBr-KYNA, 5,7-diCl-KYNA, 5-I,7-Cl-KYNA, and L-689,560 all behaved as full antagonists and were competitive with glycine. Patch-clamp recordings of cortical neurons in culture also indicated that NMDA-induced currents were antagonized by competition for the glycine site, and gave no evidence for partial agonist activity. pKi values for 5,7-diBr-KYNA and L-689,560 in these experiments were 7.2 and 7.98, respectively, similar to the affinities of these compounds in the glycine binding assay. The high affinity and selectivity of these new derivatives make them useful tools to investigate the function of the glycine site on the NMDA receptor.


Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.

The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.


Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons.

Nicotinic acetylcholine (ACh) currents were studied in cultured postnatal rat hippocampal neurons, using whole-cell voltage-clamp techniques. In most cells, ACh produces one of two types of response. One class of ACh currents exhibits rapid and profound desensitization and is sensitive to inhibition by alpha-bungarotoxin (alpha BTXN). The second class activates slowly and exhibits no desensitization during prolonged agonist applications. This slow current is insensitive to alpha BTXN. Both the fast and slow responses exhibit inwardly rectifying current-voltage relationships and pass little current at positive membrane potentials. Both currents can be recorded in the presence of 1 microM atropine but are blocked by 0.1-1.0 mM d-tubocurarine and 0.1-1.0 mM mecamylamine. These observations suggest heterogeneity of nicotinic ACh receptors in rat hippocampal neurons and provide support for functional alpha BTXN-sensitive nicotinic receptors in this region.


Common features in the interaction of tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives with the human immunodeficiency virus type 1 reverse transcriptase.

Recently, several classes of compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 replication in vitro. These include the tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and the 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) derivatives. Both the TIBO and HEPT derivatives specifically inhibit human immunodeficiency virus type 1 reverse transcriptase (RT). From a comparative study of the characteristics of RT inhibition by TIBO and HEPT, and from the competition between TIBO and HEPT for RT inhibition, we infer that both classes of compounds, although structurally unrelated, are targeted at the same site of the enzyme. Detailed functional and kinetic analyses indicate that this target site is functionally and possibly also spatially related to the substrate binding site.


GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.


Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts.

RNA editing in Trypanosoma brucei is a posttranscriptional processing event that results in the addition and deletion of uridine residues within several mitochondrial mRNAs. We have examined reactions involving pre-edited precursor RNAs in vitro. In this study, we report specific cleavage of pre-edited cytochrome b (CYb), cytochrome oxidase subunit II (COII), and cytochrome oxidase subunit III (COIII) mRNAs when incubated with T. brucei mitochondrial extracts. The pre-edited CYb RNA was cleaved near the 3'-most uridine addition sites, within the region where editing would be expected to commence. Pre-edited COII mRNA was similarly cleaved adjacent to its small editing domain, while pre-edited COIII RNA was cleaved at multiple sites in the region where uridine addition and deletion occurs in vivo. In contrast, edited versions of CYb, COII, and COIII RNAs were not cleaved within the editing domains. Such differential cleavage of the edited and pre-edited forms of these mRNAs suggests either a direct involvement in RNA editing or involvement in another aspect of mitochondrial gene expression requiring cleavage of pre-edited RNAs.


Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.


The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.


Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms.

A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.


Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.


Psoralen damage-induced plasmid recombination in Saccharomyces cerevisiae: dependence on RAD1 and RAD52.

Photoreaction with psoralen, a DNA-crosslinking reagent, induces mitotic recombination in the yeast Saccharomyces cerevisiae. Psoralen damage-induced recombination was studied with non-replicating plasmids, which transform yeast cells by undergoing recombination events with chromosomal DNA. When plasmid DNA was photoreacted with psoralen in vitro and transformed into yeast cells, transformation was stimulated by psoralen modification in a dose-dependent manner. The stimulation by psoralen damage requires RAD52 gene function and is partially dependent on RAD1. Analysis of transformants indicates that plasmid integration occurs at the homologous chromosomal loci. Multiple tandem integrations are common in repair-proficient cells, with more than 20 copies of integrated plasmid seen in some transformants. Multiple integration depends on RAD1 function; only 9% of rad1 transformants, compared to 80% of RAD transformants, contained multiple plasmid copies, while 52% of the rad1 transformants were produced by gene conversion.


Nuclease modification in Chinese hamster cells hypersensitive to DNA cross-linking agents--a model for Fanconi anemia.

Fanconi anemia is a human inherited disease that is characterized by cellular hypersensitivity to DNA cross-linking agents. A number of potential experimental models for that disorder have been developed by selecting mutants that are hypersensitive to bifunctional mutagens. The six mutants of that class in Drosophila, all of which map to the mus308 locus, express an alteration in a mitochondrial nuclease. A recent extension of that observation to cell lines from complementation group A of Fanconi anemia has established a new cellular phenotype for that disorder. In the current study an analogous enzyme has been analyzed in eight recently isolated Chinese hamster cell lines that are hypersensitive to cross-linking agents. Among these lines. V-H4 and V-B7 are shown to exhibit an enzyme modification analogous to that observed in the mutant Drosophila and human cells. These results validate the nuclease assay as an indicator of the Fanconi defect and further establish the V-H4 cell line as a valuable cellular model for analysis of the Fanconi A defect.


Quantitative detection of DNA damage in cells after exposure to ionizing radiation by means of an improved immunochemical assay.

A simple, sensitive and fast immunochemical method has been developed to quantify the amount of DNA damage in cells of human blood after in vitro exposure to ionizing radiation. The technique is based on the enhancement of the radiation-induced single-strandedness, which occurs in DNA regions flanking strand breaks, by a controlled further unwinding of the DNA in an alkaline solution. Subsequently, the DNA is attached to the wall of polystryene cups by passive adsorption. DNA damage is then quantified by determining the extent of single-strandedness with a monoclonal antibody, D1B, directed against single-stranded DNA. D1B binding is assayed with a 'second' antibody, labelled with either an enzyme or europium. The latter gives slightly more reproducible results. No radioactive labelling of DNA is required and the assay takes only 3.5 h after the collection of blood. Damage can be detected after doses as low as 0.5 Gy. The potential broader application of the method is discussed.


HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.


Determinants of cisplatin sensitivity in non-malignant non-drug-selected human T cell lines.

We have studied molecular mechanisms of cisplatin sensitivity and resistance in 3 non-malignant, non-drug-selected human T lymphocyte cell lines. HuT 78, H9, and MOLT-4 cells were assessed for sensitivity to cisplatin, DNA damage levels following defined drug exposures, drug accumulation, and DNA repair efficiency as measured by adduct removal from cellular DNA and by host-cell reactivation of cisplatin-modified plasmid DNA. Based on 3-day continuous drug exposures, the IC50 values for the cell lines were: HuT 78, 0.83 microM; H9, 0.45 microM; and MOLT-4, 0.33 microM. These cells retained this order with respect to DNA repair capability, whether measured by platinum-DNA adduct removal from cellular DNA or by host-cell reactivation assays. DNA repair values measured by these two assays were directly related to one another with a linear correlation coefficient of 0.993. At sublethal cisplatin doses the more resistant cells showed the highest levels of drug uptake. When drug uptake levels were 'corrected' for drug-induced cell kill, there were equal levels of DNA repair efficiency for a given level of drug uptake. Absolute levels of cisplatin-DNA adduct repair increased with increasing drug dose. However, at supralethal doses of drug, efficient DNA repair could be overcome in all 3 cell lines with percentage-adduct-removal dropping from a 60-80% range to a less than 30% range. We conclude that in non-malignant non-drug-selected human T cells, DNA repair appears to be the primary determinant of cisplatin sensitivity/resistance and that enhanced DNA repair may be a biologic compensatory mechanism for cells that cannot prevent cellular uptake of DNA-damaging agents.


Transcription-dependent and independent DNA excision repair pathways in human cells.

alpha-Amanitin, an inhibitor of RNA polymerase II, has little effect on either UV-induced incision or repair synthesis in cultured normal human fibroblasts but almost completely inhibits both processes in xeroderma pigmentosum group C fibroblasts. Cycloheximide, at a concentration which inhibits protein synthesis by 75-80%, has no effect on incision or repair synthesis in either cell type, which argues that the effects of alpha-amanitin on repair occur at the level of transcription. Cot analysis demonstrates that UV-induced repair synthesis occurs at similar levels in highly repetitive, middle repetitive and single copy sequence in both normal and xeroderma group C cells. We conclude that normal cells must have at least two excision repair pathways for repair of UV-induced damage, one dependent on transcription and the other independent.


Differences in DNA damage induced by mutagenic and nonmutagenic 4-aminoazobenzene derivatives in Escherichia coli.

DNA lesions produced in Escherichia coli AB2500 (uvrA) exposed to the carcinogen N-hydroxy-3-methoxy-4-aminoazobenzene (N-OH-3-MeO-AAB) or the noncarcinogen N-hydroxy-2-methoxy-4-aminoazobenzene (N-OH-2-MeO-AAB) were investigated by alkaline sucrose gradient sedimentation and 32P-postlabeling analysis. Alkali-labile sites appeared to be formed equally in cells treated with both aminoazobenzene derivatives. 32P-Postlabeling analysis revealed that the 3-MeO-AAB-DNA adduct level was 25-fold higher than that for 2-MeO-AAB-DNA adducts. In addition to major adducts, 4 minor spots were detected in N-OH-3-MeO-AAB-treated cells, while only one major adduct was found in N-OH-2-MeO-AAB-treated cells. The mutagenicities and cytotoxicities were also determined with E. coli with different repair capacities; we found that repair of 3-MeO-AAB damages is strongly dependent on the UVR repair system. Moreover, N-OH-3-MeO-AAB, but not N-OH-2-MeO-AAB, could induce recA and umuC gene expression, which was higher in uvrA strains than in the wild type.


Investigation of the mutagenic activity in Salmonella typhimurium of the furochromone khellin, proposed as a therapeutic agent for skin diseases.

The photomutagenicity of the furochromone khellin was tested in Ames Salmonella strains using 8-methoxypsoralen (8-MOP) and 4,5', 8-trimethylpsoralen (TMP) as positive controls. When khellin was assayed with strain TA1537, mutation induction was not detectable; in the same strain, an equitoxic dose (52-56% level of survival) of TMP (used at a concentration 12-fold lower than khellin and with a UVA dose 83-fold lower than that used with khellin) yielded an increase in revertants/plate 3-fold above the spontaneous background. In strain TA102, khellin plus UVA treatment yielded a 2-fold increase in revertants/plate above the spontaneous background (79% survival). 8-MOP, however, used at a concentration 8-fold lower than khellin with a UVA dose 13-fold lower than khellin, yielded an increase in revertants/plate about 14-fold above background (66% survival) in the same strain. These data show that khellin has a weak photomutagenic potential and, along with the previously reported low photogenotoxic potential in eukaryotic cell systems, support the notion that khellin may be safer than bifunctional psoralens for clinical use.


Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.


Sister-chromatid exchanges in operating room personnel.

Sister-chromatid exchange (SCE) analysis was carried out in 67 operating room personnel (anaesthetists M.D.; anaesthesia nurses and anaesthesia unit technicians) exposed to waste anaesthetic gases such as halothane, nitrous oxide and isoflurane and in 50 healthy unexposed controls. The SCE frequencies were increased significantly in operating room personnel as compared to controls. A significant increase in SCEs was found in non-smoking operating room personnel as compared to non-smoking controls. This study supports the existence of an association between occupational exposure to mutagens and an increase in SCEs in lymphocytes.


Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5.

Effects of p-aminobenzoic acid (PABA) and of 4-[(2-oxo-3-bornylidene)methyl]-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems. A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci. The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination. The induction of forward mutations was measured in Chinese hamster V79 cells. Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5. V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5. PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations. At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.


Biomonitoring of genotoxic exposure among stainless steel welders.

A biosurvey in the Danish metal industry measured the genotoxic exposure from stainless steel welding. The study comprised measurements of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), unscheduled DNA synthesis (UDS) in peripheral lymphocytes and serum immunoglobulin G. Environmental monitoring of welding fumes and selected metal oxides, biomonitoring of chromium and nickel in serum and urine and mutagenic activity in urine, and evaluation of semen quality were also done. Manual metal arc (MMA) welding and tungsten inert gas (TIG) welding were the dominant welding processes. A higher frequency of chromosomal aberrations, classified as translocations, double minutes, exchanges and rings, was observed in stainless steel welders than in non-welders. SCE was lower in welders working with both MMA and TIG welding than in reference persons. N-Acetoxy-N-acetylaminofluorene (NA-AAF)-induced UDS was lower in 23 never-smoking welders than in 19 unexposed never-smokers. Smoking was a confounding factor resulting in significantly higher CA, SCE, NA-AAF binding to DNA and mutagenic activity in urine. Age was also a confounder: CA, SCE, NA-AAF binding to DNA and UDS increased significantly with age. No significant correlation between SCE and CA or between CA and UDS was found. UDS decreased significantly with increasing lymphocyte count and a higher lymphocyte count was seen in MMA welders than in reference persons and in smokers than in non-smokers. Differences in the composition among lymphocytes in exposed persons compared with non-exposed are suggested. MMA welding gave the highest exposure to chromium, an increased number of chromosomal aberrations and a decrease in SCE when compared with TIG welding. Consequently improvements in the occupational practice of stainless steel welding with MMA is recommended.


Frequencies of chromosomal aberrations in pesticide sprayers working in plastic green houses.

The frequency of chromosome aberrations was studied in peripheral blood lymphocytes of 29 workers occupationally exposed to a mixture of pesticides and in 14 age- and sex-matched healthy controls. There was a significant increase in chromosome aberrations in sprayers when compared to unexposed persons (2.39% compared to 0.54%). No positive correlation between the frequency of chromosome aberrations and the duration of exposure was observed. No significant difference between smokers and non-smokers was found.


Sister-chromatid exchanges induced by vinyl esters and respective carboxylic acids in cultured human lymphocytes.

Vinyl acetate--an efficient inducer of sister-chromatid exchanges (SCEs)--is known to be hydrolyzed in mammalian cells into acetic acid and acetaldehyde, the latter being the likely metabolite responsible for the SCE induction. As similar hydrolysis to acetaldehyde and to a carboxylic acid is also expected for other vinyl esters, five such compounds--vinyl formate, vinyl chloroformate, vinyl propionate, vinyl crotonate and vinyl-2-ethylhexanoate--and five carboxylic acids--formic acid, acetic acid, propionic acid, crotonic acid and 2-ethylhexanoic acid--were tested for their ability to induce SCEs in cultured (72 h) human lymphocytes with a 48-h treatment, starting at 24 h after culture initiation. Vinyl formate, vinyl propionate and vinyl crotonate induced a clear dose-dependent increase in the number of SCEs/cell at concentrations of 0.125-0.5 mM and vinyl chloroformate at 0.063-1 mM, i.e., at roughly the same concentration range as vinyl acetate and acetaldehyde. Vinyl-2-ethylhexanoate required slightly higher concentrations (0.25-4 mM) for SCE induction. All of the carboxylic acids tested also elevated SCEs, but only slightly. Formic acid and crotonic acid produced some SCE increase at a concentration of 10 mM, acetic acid at 5 and 10 mM and propionic acid at 2.5 mM. 2-Ethylhexanoic acid induced SCEs at a lower concentration range (0.63-2.5 mM) than the other acids. The positive concentrations of the first three carboxylic acids lowered the pH of the culture medium immediately after the treatment by 0.5-1.0 pH unit (lowest observed pH 6.53). The pH differences from the control cultures became smaller in measurements done 24 h and 48 h after the beginning of treatment. Propionic acid and 2-ethylhexanoic acid affected medium pH only slightly (maximum drop 0.2 pH units) at the concentrations that induced SCEs. The results lend support to the idea that the efficient SCE induction observed with the vinyl esters results from the formation of acetaldehyde, with carboxylic acids--with the possible exception of 2-ethylhexanoic acid--playing no significant role. The slight SCE induction obtained with the carboxylic acids cannot be explained by lowered pH alone.


Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.


Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) requires metabolic activation to express its carcinogenic activity. This activation leads to the formation of methylating and pyridyloxobutylating agents. To determine the possible biological effects mediated by each of these metabolic pathways we have studied the activities of model compounds that are metabolized to either a methylating or pyridyloxobutylating species. Each model compound was evaluated for its mutagenic activity in both prokaryotic and eukaryotic cell systems. The model compounds were also tested for their ability to induce asynchronous replication of viral DNA sequences. We demonstrate here that both the methylating model compound acetoxymethylmethylnitrosamine (AMMN) and the pyridyloxobutylating model compound 4-(acetoxymethyl)-1-(3-pyridyl)-1-butanone (NNKOAc) were mutagenic in strains TA98, TA100, and TA1535 but not TA102. While NNKOAc appeared to be 10 times more potent than AMMN in Salmonella, AMMN was found to be a more potent mutagen in mammalian G12 cells. Both chemicals could induce asynchronous replication of polyoma DNA sequences in rat fibroblast cells carrying an integrated copy of the polyoma virus with AMMN appearing to be more active. Measurement of DNA adduct levels suggest that the damage produced by NNKOAc was at least as active as that produced by AMMN when viewed on a per adduct basis. The possible implications of the biological activities exhibited by methylating and pyridyloxobutylating model compounds to NNK induced carcinogenesis are discussed.


Irreversible association of peptides with class II MHC molecules in living cells.

Functional, morphological and biochemical evidence indicates that class II major histocompatibility complex (MHC) molecules associate with processed peptides during biosynthesis. Peptide/MHC complexes in living cells have been reported to be less stable than similar complexes generated in vitro, which has led to the suggestion that there may be a peptide exchange mechanism operating in vivo. Although this could increase the capacity for binding incoming antigens, it would reduce the efficacy of processed antigenic peptides by exchanging these for self peptides. Here we measure the half-life of peptide/class II complexes in human antigen-presenting cells and find that it is very similar to the half-life of class II molecules themselves, indicating that peptides are bound irreversibly under physiological conditions. Thus class II MHC retains long-term 'memory' of past encounters with antigen to maximize the opportunity for T cell/antigen-presenting cell interaction.


Caudal ventrolateral medullary projections to the nucleus of the solitary tract in the cat.

The projections of neurons, in and around the A1 noradrenergic cell group of the caudal ventrolateral medulla (VLM), to nucleus of the solitary tract (NTS) were studied in the cat using the anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L). PHA-L was micro-iontophoresed into the region of the A1 noradrenergic cell group and after a 7-17 day survival period animals were sacrificed and brainstem sections were processed for PHA-L or tyrosine hydroxylase (TH) immunoreactivity. PHA-L injections within the region of the A1 cell group resulted in labelled fibers with their presumptive terminal boutons primarily in the ipsilateral commissural and medial subnuclei of NTS. A light projection to the ipsilateral parvocellular lateral and ventrolateral subnuclei of the NTS complex was also observed. These data demonstrate that neurons in the region of the A1 noradrenergic cell group project to regions of NTS that receive cardiovascular afferent inputs and suggest that VLM may influence the activity of neurons in NTS involved in the reflex regulation of the circulation.


Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons projecting from the midbrain periaqueductal gray to the nucleus tractus solitarii in the rat.

Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) were observed to send their axons to the nucleus tractus solitarii in the rat by the retrograde horseradish peroxidase tracing method combined with the immunocytochemical technique. These neurons were most frequently observed in the ventrolateral subnucleus and ventral portion of the medial subnucleus of the PAG at the entire rostrocaudal levels.


The cGMP-gated channel of rod outer segments is not localized in bipolar cells of the mammalian retina.

It has recently been suggested (Nature, 346 (1990) 269-271) that ON-bipolar cells express the same biochemical cascade and guanosine 3',5'-cyclic monophosphate (cGMP)-gated cation channel as rod outer segments. An antibody directed against the cGMP-gated channel of bovine rod outer segments was applied to cryostat sections of rat and cat retinae. No immunocytochemical labelling was found in bipolar cells. Therefore, if those cells express a cGMP-gated channel, it must be immunologically different to the 63 kDa protein constituting the cGMP-gated channel of the outer segment.


High and low affinity membrane binding sites for fibroblast growth factors in the developing chick brain.

Acidic and basic fibroblast growth factors (aFGF and bFGF), two mitogenic, neurotrophic and angiogenic molecules, are present in the embryonic chick brain but their function remains unclear. In order to approach the biological activity of FGFs during brain development, we have looked for their receptors and studied their regulation through chick brain development. Competitive binding studies realized on brain membranes indicated the presence of two classes of FGF binding sites: high affinity binding sites (dissociation constant, Kd = 100 pM) and low affinity binding sites (Kd = 20 nM). Cross-competition experiments show that these two classes of binding sites both interact with aFGF and bFGF. The number of sites in these two classes of binding sites changes during embryogenesis. On the one hand, the membrane capacity of high affinity sites decreases from E7 (1 +/- 0.2 pmol/mg of protein) to E15 (0.5 +/- 0.2 pmol/mg of protein); on the other hand, the membrane capacity of low affinity sites increases from E15 (25 +/- 4 pmol/mg of protein) to P1 (75 +/- 20 pmol/mg of protein). Cross-linking experiments revealed the presence of two putative receptor forms of molecular masses of about 130 and 95 kDa. These results suggest that the biological activity of aFGF and bFGF during brain embryogenesis could be regulated by the expression of high and low affinity binding sites for these growth factors.


Apolipoprotein B immunoreactivity in senile plaque and vascular amyloids and neurofibrillary tangles in the brains of patients with Alzheimer's disease.

Based upon our previous finding of the association of apolipoprotein E (apoE) immunoreactivity with cerebral amyloids and neurofibrillary tangles (NFTs), we examined immunohistochemically whether this is also the case for apolipoprotein B (apoB). Polyclonal antibody to apoB immunosustained senile plaque amyloid, vascular amyloid, subpial amyloid deposits and intracellular NFTs in formalin-fixed, paraffin-embedded brain sections from patients with Alzheimer disease. Hydrated autoclave pretreatment of the sections enhanced the staining of plaque amyloid. The results may suggest a role of apoB in amyloid and NFT formation.


Infections involving implanted cardioverter defibrillator devices.

Infection is one of the most serious complications in patients with an implanted cardioverter defibrillator. The diagnosis is facilitated by computed tomographic and radionuclide imaging. Infection may be caused by intraoperative contamination or hematogenous seeding. In view of the serious consequences, the emphasis should be on prevention of these events. Perioperative antibiotic prophylaxis is common practice but the utility of prophylactic antibiotic remote from surgery is questionable. Strict adherence to asepsis and a meticulous surgical technique are essential. Identification of risk factors in the individual patient allows a patient-tailored treatment policy that may add to infection prevention. If implant infection does occur, complete removal of the system is most successful.


Role of ventricular tachycardia surgery and catheter ablation as complements or alternatives to the implantable cardioverter defibrillator in the 1990s.

Although the implantable cardioverter defibrillator is used increasingly, other nonpharmacological approaches have their indications and merits. Furthermore, as the natural history of ventricular tachyarrhythmias or their underlying structural cardiac abnormality, i.e., coronary artery disease, dilated cardiomyopathy, arrhythmogenic right ventricular disease, etc. change, the mode of therapy may be modified accordingly. Because of the disappointing results of the CAST study in previously asymptomatic patients after myocardial infarction and the evidence that failure of one or two antiarrhythmic drugs tested by programmed ventricular stimulation in patients with documented sustained ventricular tachycardia or fibrillation predicts further drug failure, there will be a significant increase in the use of implantable cardioverters defibrillators in the 1990s. However, care should be taken to avoid inappropriate use of these devices.


Principles of radiation therapy in the treatment of bone metastases.

The radiation oncologist is an important member of the multidisciplinary team involved in the management of patients with metastatic bone disease. Radiation therapy provides local tumor control of the metastatic deposit and effective pain relief in the majority of patients. If there is a risk of fracture, prophylactic fixation is done before radiation therapy.


Postnatal placental confirmation of trisomy 2 and trisomy 16 detected at chorionic villus sampling: a possible association with intrauterine growth retardation and elevated maternal serum alpha-fetoprotein.

Detection of trisomy 2 and trisomy 16 mosaicism through chorionic villus sampling (CVS) is not an infrequent finding. We describe here two cases, one of non-mosaic trisomy 2 and the other of high level mosaicism for trisomy 16. Amniocentesis in both cases demonstrated non-mosaic 46,XY karyotypes. Each pregnancy continued to delivery of liveborn, normal-appearing boys; both pregnancies were complicated by severe intrauterine growth retardation (IUGR). Postnatal studies of placental biopsies in both cases confirmed the original CVS findings, whereas cord blood karyotypes were normal in both boys. Both children have demonstrated adequate catch-up growth.


The psychological effects of false-positive results in prenatal screening for fetal abnormality: a prospective study.

The purpose of the study was to describe the impact of false-positive results from initial maternal serum alpha-fetoprotein (MS-AFP) screening. The analyses compared two groups of women, those receiving a negative result (n = 346) and those receiving an initial positive result (n = 26), over four time points--prior to testing, immediately after testing, later in pregnancy, and in the post-partum period. Receiving an abnormal result was associated with high levels of anxiety which were reflected in increased worry about the baby's health and a more negative attitude towards the pregnancy and the baby. Women who had an initial abnormal result were offered a variety of further tests. Those women who went on to have amniocentesis were less worried about their baby's health in the third trimester and also less anxious post-partum than those who did not have amniocentesis. In view of the increasing number of screening tests available, it is necessary to establish whether and how these levels of distress can best be reduced.


CA-125 as a maternal serum marker for Down's syndrome in the first and second trimesters.

CA-125, alpha-fetoprotein (AFP), and human chorionic gonadotropin (HCG) were determined in maternal serum in the first trimester from 14 women with a Down's syndrome fetus and 61 women with a healthy fetus. In the second trimester, 15 and 60 serum samples were determined from women with a Down's syndrome and a healthy fetus respectively. In both trimesters, maternal serum CA-125 was found to be elevated in Down's syndrome pregnancies compared with controls. Using discrimination functions, our preliminary results indicate that CA-125 is a better marker than AFP and HCG respectively for a Down's syndrome fetus in the first trimester and improves the detection rate in the second trimester.


Trypanosoma cruzi: enhanced alpha-macroglobulin levels correlate with the resistance of BALB/cj mice to acute infection.

Trypanosoma cruzi proteinases are very likely involved in host-cell invasion. Physiological plasma-proteinase inhibitors from the macroglobulin (MG) family, among them alpha-2-macroglobulin (A2M), are found in tissues and in the plasma of mammals. By complexing to all classes of proteinases, MGs inhibit their action on high-molecular-weight substrates. In vitro studies have shown that A2M impairs T. cruzi proteases and, consequently, the parasite's ability to invade host cells and enhances the phagocytic and microbicidal actions of resident macrophages against T. cruzi. To test the hypothesis of a putative "protective" effect for MG, we quantified it in BALB/cj mice during the course of an experimental T. cruzi infection, comparing a posteriori the levels in mice that died with those in animals that survived, which were considered as being susceptible and resistant to the infection, respectively. The results showed that surviving mice showed an increase in plasma concentrations of MG during the first few weeks after the infection, whereas the levels in mice that died during the acute phase did not differ significantly from those in non-infected mice. These findings and the previous in vitro data indicate a role for physiological proteinase inhibitors, particularly alpha-macroglobulins, in resistance to T. cruzi infection, whereby a balance between parasite proteases and host protease inhibitors may be crucial. MG may thus participate in the complex network of reactions involved in the early acute phase of the disease and contribute by conferring to the host an ability to survive the infection.


Extrinsic control of the release of galanin and VIP from intrinsic nerves of isolated, perfused, porcine ileum.

By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.