GM-CSF and G-CSF stimulate the synthesis of histamine and putrescine in the hematopoietic organs in vivo. Histamine and putrescine (a precursor of polyamines) are formed by histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), respectively. Within a few hours after injection of a lipopolysaccharide (LPS) into mice, HDC is induced in the liver, spleen, lung and bone marrow, and ODC is induced in the liver, spleen and bone marrow. Since LPS is known to stimulate the production of various cytokines, the abilities of various cytokines to induce HDC and ODC in the tissues of mice were examined. IL-2, IL-6, IL-8, IFN gamma and M-CSF were ineffective. IL-1 alpha, IL-1 beta, TNF alpha and TNF beta induced HDC and ODC, as does LPS. On the other hand, GM-CSF and G-CSF induced HDC and ODC only in the spleen and bone marrow within a few hours after their injection. These results suggest that, in addition to their roles in inflammation or immune responses, HDC and ODC are also involved in an early stage of hematopoiesis.


The antigen-specific induction of normal human lymphocytes in vitro is down-regulated by a conserved HIV p24 epitope. Synthetic peptides containing amino acid sequence 218-238 of the core protein p24 of human immunodeficiency virus type 1 (HIV-1) and progressively shorter sequences at its C-terminus, were tested for their effect on antigen dependent in vitro responses of peripheral blood lymphocytes (PBL) from normal human donors. A peptide as short as 7 amino acids, corresponding to a highly conserved sequence, was able to inhibit in a dose-dependent manner the induction of a specific primary antibody response to the sheep red cell (SRC) antigen, as well as the proliferative response to recall microbial antigens. The results of this study constitute additional evidence of the immunoinhibitory effects of HIV components and may help to unravel some of the pathogenic mechanisms of AIDS. Moreover, they are of potential relevance for the development of immunoprophylactic and therapeutic strategies.


Localization of an immunoinhibitory epitope of the cysteine proteinase, cathepsin L. Antibodies, raised in chickens (IgY) and rabbits (IgG) against the lysosomal proteinase cathepsin L, targeted the enzyme in an ELISA and Western blot. In contrast to the rabbit IgG, the chicken IgY was immunoinhibitory towards cathepsin L. An epitope that elicits immunoinhibitory antibodies has been localized to an active site-associated peptide sequence. The corresponding free peptide, coated down in an ELISA, is recognised by the chicken IgY, but not the rabbit IgG. This peptide was able to inhibit the immunoinhibition of cathepsin L by chicken anti-cathepsin L IgY, suggesting its complete or partial identity with an immunogenic epitope for chickens in whole cathepsin L.


Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using biotinylated antigen. A generally applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen the method was applied to the determination of epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody were then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxidase. The method used here alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs, and is convenient for mapping analysis of many MAbs if the corresponding purified antigen is available.


Palliative segment III biliary bypass (left cholangio-jejunostomy) in malignant block at porta hepatis. Segment III cholangio-enteric anastomosis was performed in 17 patients with obstructive jaundice due to unresectable malignancies at the porta hepatis. The operative mortality was 6% (1/17) and morbidity 30% (5/17). More than 50% fall in bilirubin level with symptomatic improvement in pruritus was seen in 13 patients. Three patients had 25%-50% fall in bilirubin level. This procedure is safe and effective in palliation of unresectable hilar obstruction.


Increased transcripts for B-type natriuretic peptide in spontaneously hypertensive rats. Quantitative polymerase chain reaction for atrial and brain natriuretic peptide transcripts. The cardiac natriuretic peptide family includes atrial natriuretic factor and brain or B-type natriuretic peptide, also known as iso-atrial natriuretic factor (isoANF). Although these peptides contribute to cardiovascular homeostasis, their respective roles remain unclear. To study regulation of atrial natriuretic factor and isoANF gene expression during progression of hypertension, we developed a quantitative polymerase chain reaction protocol to measure their transcript level in spontaneously hypertensive rat (SHR) hearts. At the onset of hypertension, atrial natriuretic factor transcripts in 5-week-old SHR were 50% of those of age-matched Wistar-Kyoto (WKY) rats, whereas the level of isoANF transcripts was similar in atria and twofold higher in ventricles. Because atria are the major sites of atrial natriuretic factor gene expression and ventricles contribute predominantly to cardiac isoANF synthesis, total atrial natriuretic factor messenger RNA (mRNA) in the hearts of 5-week-old SHR was about 50% of that in WKY rats, and total isoANF mRNA content was already higher than in control rats. In left ventricles and ventricular septa, progression of hypertension led to a maximal increase of twofold and fourfold in atrial natriuretic factor and isoANF mRNA levels, respectively, with no detectable change in right ventricles. In the atria of older SHR, atrial natriuretic factor and isoANF mRNA levels were comparable to those of age-matched controls. These data indicate that, although increased blood pressure stimulates both atrial natriuretic factor and isoANF gene expression, regulation of the two natriuretic peptide genes is not temporally coordinated in all cardiac compartments. Furthermore, isoANF mRNA is already induced in the ventricles at the onset of the hypertensive stage, and in older SHR, the isoANF gene is hyperresponsive to progression of hypertension compared with atrial natriuretic factor. Thus, isoANF might represent a very sensitive marker of cardiac changes in hypertension.


Transthyretin is an inhibitor of monocyte and endothelial cell interleukin-1 production. Human serum was found to contain an inhibitor of constitutive interleukin-1 (IL-1) production by human umbilical vein endothelial cells (ECs). Purification of the serum activity by anion exchange chromatography, molecular sieve HPLC, and hydroxyl apatite chromatography yielded material 82% pure with a molecular weight of 17 kDa by SDS-PAGE. Amino acid sequencing revealed the purified inhibitor to be transthyretin (TTR), a liver-derived protein. There was a 42.6% reduction in the production of spontaneous IL-1 activity in EC supernatants after coculture with 10 micrograms/ml TTR. TTR was subsequently found by ELISA to inhibit LPS-stimulated IL-1 production by cells of the human monocytic leukemia line THP-1 by 47.1 +/- 9.4%, whereas a less striking but still significant inhibition of monocyte-derived IL-1 beta production was also observed. Inhibition of IL-1 secretion correlated with increased IL-1 mRNA synthesis in both THP-1 cells and monocytes. Furthermore, TTR was associated with increased intracellular concentrations of IL-1 beta. These data suggest that TTR functions by inhibiting processing of newly synthesized peptide for secretion. This novel inhibitory effect of TTR on the production of IL-1 activity suggests a previously unrecognized endogenous antiinflammatory mediator.


Effect of substance P and somatostatin on migration of polymorphonuclear (PMN) cells in vitro. The effects of the two neuropeptides, substance P (SP) and somatostatin (SOM), on the migration of polymorphonuclear cells derived from 13 volunteers were investigated. The neuropeptides were applied in concentrations between 10(-12) and 10(-6) M. Only at a concentration of 10(-6) M SP did the chemotaxis of PMN cells increase slightly but statistically significantly. In contrast to SP, SOM showed a significant dose-dependent stimulation of chemotaxis, which was first traceable at 10(-10) M and increased up to 10(-6) M. Although it is uncertain whether in vivo SP and SOM contribute directly to the invasion of PMN cells into the joint cavity, the influence of these neuropeptides on PMN migration in vitro is a further indication of the neuropeptide involvement in the genesis of inflammation.


Expression of different tenascin isoforms in normal, hyperplastic and neoplastic human breast tissues. Functionally different tenascin (TN) isoforms, containing varying numbers of a 91 amino-acid motif resembling the fibronectin type-III homology repeat, may be generated by alternative splicing of the TN primary transcript. In fact, only the TN isoform containing the alternatively spliced region can induce loss of focal adhesion in cultured cells and seems to be able to facilitate cell migration. We examined the patterns of alternative splicing of the TN primary transcript in normal, hyperplastic and neoplastic breast tissues, and found that, in all the invasive breast carcinomas analyzed, the relative amount of TN mRNA in which the alternatively spliced region was included was about 10 times higher than in RNA from normal breast tissues. A similar result was observed in phyllodes tumors and in those fibroadenomas which showed very high stromal cellularity. Western-blot analysis using different monoclonal antibodies showed the same pattern as that seen in Northern blotting. The data reported here suggest that, in the breast, expression of the high-molecular-mass TN isoform is a marker of stromal element proliferation and that, in invasive breast carcinomas, this TN isoform could play a role in generating a permissive environment for proliferation, invasion and metastasis of neoplastic epithelial cells.


Breast cancer is associated with loss of the c-kit oncogene product. The proto-oncogene c-kit encodes a tyrosine kinase receptor related to the PDGF/CSF-1 receptors. Mutations of this gene result in impairment of hematopoiesis, melanogenesis and gametogenesis. Using monoclonal antibodies to the c-kit gene product, we have analyzed its expression in normal and transformed human tissues. Unexpectedly, the receptor was found to be expressed in normal mammary epithelium. While in benign breast lesions, the c-kit gene product was detected at variable levels in 82% of the instances, in primary tumors, no product could be identified in 87% of the cases. This phenotype is maintained in metastatic foci. These findings were confirmed by paired Northern blot analysis of RNA preparations from normal and tumor tissues. These results demonstrate that the c-kit receptor may also be involved in the growth control of mammary epithelium and that this function may be impaired following malignant transformation and de-differentiation.


Infrequent detection of HIV-1 components in tears compared to blood of HIV-1-infected persons. Beside the risk of infection via HIV-1-contaminated blood, ophthalmologists are especially interested in the possibility of HIV-1 infection via tears. Therefore we tried to isolate HIV-1 from tears of 50 HIV-1-infected persons in different stages of disease by reverse transcriptase (RT) and by p24-antigen (p24-AG) in the cultures. Simultaneously we tried to isolate HIV-1 in the supernatant from peripheral blood lymphocytes (PBL), which was successful in 32 of the 50 examined specimens. HIV-1 could not be isolated from the tears of these persons. In addition, polymerasechain-reaction (PCR) was performed to detect proviral sequences (gag, pol, env) of HIV-1 in tears and blood of ten HIV-1-infected patients. While in all the examined patients gag, pol and env could be detected in the blood samples, only one tear sample was found positive for gag and pol DNA fragments. These results indicate that tears of HIV-1-positive contain extremely low quantities of tissue culture infectious doses (TCID) of HIV-1 in contrast to PBL. HIV-1 infection via tears therefore appears to be unlikely.


Secretion of cell-adhesion-promoting factors, fibronectin, fibronectin fragments and a 53-kDa protein, by human rectal adenocarcinoma cells. Production of cell-adhesion proteins was examined in 10 cell lines and 5 cultured human cancer cells at an early passage. Two-thirds of the tested cells produced and secreted into their culture medium variable amounts of material active in promoting cell attachment. One of the rectal carcinoma cell lines, CaR-I, grew well in serum-free medium and secreted a large amount of the active principle. The active principles produced by CaR-I cells were characterized after partial purification, and were found to be fibronectin and its fragments. The presence of fibronectin and its fragments was proved by the following facts: (1) reactivity to the monoclonal antibodies which recognize different epitopes of fibronectin, and (2) reactivity to RGD peptide which is the attachment sequence of fibronectin. In addition to fibronectin and its fragments, CaR-I cells were also shown to produce a 53-kDa attachment factor. Unexpectedly, the protein was proved to be most probably the p53 suppressor gene product.


Atrial premature beats coupling interval determines lone paroxysmal atrial fibrillation onset. In 20 patients with recurrent episodes of lone paroxysmal atrial fibrillation we assessed the onset pattern of each episode of either atrial fibrillation or of atrial flutter during a 24-h Holter monitoring. We evaluated 24 twenty-four-hour Holter tape recordings and our data are related to 168 episodes of paroxysmal atrial fibrillation and 27 episodes of paroxysmal atrial flutter. Eighty-five percent of atrial fibrillations and 67% of atrial flutters were of short duration (less than 5 min). The majority of patients (80%) had either nocturnal or daily episodes of arrhythmia and PP intervals immediately before onset of arrhythmia did not show significant variations in 77% of cases. The coupling interval of the supraventricular premature beats eliciting atrial fibrillation was significantly shorter than the coupling intervals of the spontaneous isolated supraventricular premature beats (p less than 0.0001); again, in 6 patients with either atrial fibrillation or flutter, the coupling interval at onset of fibrillation was significantly shorter in comparison to flutter (p less than 0.0001). In conclusion, vagal or sympathetic prevalence does not seem to influence significantly the beginning of the arrhythmia, while the coupling interval of the atrial premature beats plays a critical role in the inducibility of atrial flutter or fibrillation.


Effect of immunosuppressive agents on the guanethidine-induced sympathectomy in athymic and euthymic rats. Guanethidine sulphate causes destruction of peripheral sympathetic neurons and infiltration of mononuclear inflammatory cells in the sympathetic ganglia of both athymic nude (rnu/rnu) and euthymic LEW/Mol rats. The effect of guanethidine is believed to be an autoimmune reaction. To determine the effect of immunosuppressive drugs concurrently with guanethidine treatment both athymic and euthymic rats were treated with guanethidine 40 mg/kg i.p. daily for 14 days, cyclophosphamide 100 mg/kg i.p. on days 1 and 8, methylprednisolone 10 mg/kg and cyclosporin A 10 mg/kg daily from days 1 to 7, and then every other day from days 8 to 14. The number of neurons in the sympathetic ganglia was counted and four subpopulations of mononuclear inflammatory cells were identified by monoclonal antibodies MHC II, CD8 T-cells/NK-cells, CD5 T-cells, CD4 T-cells/macrophages. Our results show that the immunosuppressive drugs used were unable to prevent the guanethidine-induced reduction of sympathetic neurons, although the number, of neurons following guanethidine-methylprednisolone treatment was significantly higher compared with guanethidine alone in both athymic and euthymic rats. The identification of mononuclear cells in the sympathetic ganglia showed that the CD8/NK and CD5 populations were the populations primarily responding to guanethidine treatment. Both CD8/NK and CD5 populations were absent without guanethidine, but increased significantly following guanethidine in both athymic and euthymic animals. None of the immunosuppressive drugs used could prevent the guanethidine-induced rise in the CD8/NK population in neither athymic nor in euthymic rats. The rise in the CD5 population was suppressed following treatment with all immunosuppressive drugs in athymic rats, but only following methylprednisolone in euthymic animals. These results indicate that guanethidine induces proliferation of T-cells in euthymic rats and non-functional CD5 positive pre T-cells in athymic animals. The CD5 population in both athymic and euthymic animals appears relatively more sensitive to immunosuppressive drugs than the NK-cell population also activated by guanethidine. This relatively resistant NK-cell population seems to play an important role in the guanethidine-induced destruction of sympathetic neurons and can explain why the guanethidine-induced immunological reaction could not be fully prevented by the immunosuppressive drugs used. The conclusion is that guanethidine induces destruction of sympathetic neurons by a NK-cell-mediated reaction.


Relapsing fever and its serological discrimination from Lyme borreliosis. Patients with Borrelia-caused relapsing fever produce cross-reacting antibodies to Borrelia burgdorferi, the anti-genetically related causative agent of Lyme borreliosis. The antibody response of the serum of a patient (acute and convalescent) with relapsing fever was analysed by the immunoblot technique using Borrelia hermsii and B. burgdorferi as antigens. The diagnosis was established by microscopic detection of spirochetes in the patient's blood. The patient's serum showed significantly elevated titers of IgG and IgM in a B. burgdorferi indirect immunofluorescence assay. Immunoblot analysis indicated the presence of cross-reacting antibodies directed to B. burgdorferi antigens with apparent molecular weights of 60, 41, 40, 36, 30 and 20 kDa.


Role of adhesion molecules in the immune reaction to M-MSV-induced tumors. We have investigated the in vivo role of 2 different adhesion molecules, LFA-1 and LECAM-1, in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which undergo a peculiar spontaneous regression due to generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated administration of anti-LFA-1 monoclonal antibody (FD441.8 MAb), i.p. or at the site of virus inoculation, enhanced tumor growth and delayed regression, while i.p. administration of anti-LECAM-1 MEL-14 MAb gave rise to tumors that grew progressively and caused host death. Evaluation of the immunological response in MAb-treated mice showed reduced generation of virus-specific CTL precursors (p) in the spleen of animals given FD441.8 MAb i.p.; CTLp frequency in locally treated mice overlapped with that of control mice injected with virus only. FD441.8 MAb treatment did not interfere with CTL homing in the tumor, since the frequency of M-MSV-specific CTLps in sarcomas was similar in treated and control mice. Cytofluorimetric analysis indicated that the majority of tumor-infiltrating lymphocytes (TIL) from MAb-treated mice were covered by anti-LFA-1 MAb, and lacked cytotoxic activity when assayed against target cells bearing relevant tumor antigens. Instead, in mice injected i.p. with MEL-14 MAb, a very low frequency of CTLps was detected in lymph nodes draining the tumor area, and within the tumor. Our results indicate that enhanced tumor growth, depending on the MAb used, is the resultant of an inhibitory effect on different T-lymphocyte functions. Tumor progression in anti-LFA-1 MAb-injected mice is explained mostly by blockage of CTL lytic activity at the tumor site; in mice receiving i.p. MEL-14 MAb treatment, by the failure of naive T lymphocytes to enter peripheral lymph nodes and subsequently by the lack of generation of tumor-specific CTLs.


The influence of oral and subcutaneous administration of a mixed beta-adrenergic agonist on tissue and cell growth. The non-selective beta-adrenergic agonist, metaproterenol, administered orally (2 ppm and 20 ppm) and subcutaneously (0.1 mg/Kg/d) for 21 days to young male rats induced no changes in animal growth or food intake. However, the rats treated with metaproterenol showed increased muscle gastrocnemius weight, which was accompanied by a muscle hyperplasia (DNA content increase) and a reduction in cellular size (protein/DNA ratio decrease). The beta-agonist did not affect liver growth or liver cellularity when assessed through the nucleic acid content. The increase in muscle mass was attributed, at least in part, to a reduction in protein breakdown as muscular cathepsin A activity was significantly lower in beta-agonist treated rats.


Sequence homology between two immunodiagnostic fusion proteins from Echinococcus multilocularis. Serological diagnosis of alveolar hydatid disease using crude parasite antigen is problematical with the result that several groups have addressed the question of specific serodiagnosis using defined native or recombinant antigens. Sequence data are available for two lambda gt 11 cDNA clones, designated EM4 and pEM10, which express E. multilocularis species-specific proteins in immunodiagnostic assays using human sera. Here, we have drawn attention to the extensive similarities between these antigens and have compared their characteristics since both may prove valuable in the future diagnosis of alveolar hydatidosis.


Structure activity of C-terminal modified analogs of Ac-CCK-7. Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.


Proteinase-catalyzed conversion of a substance P-precursor peptide. The protease-catalyzed conversion of peptides and proteins produced by recombinant DNA technology is a promising method for large-scale production of peptides including those with non-proteinogenic structural elements. As a model system we have investigated the proteinase-catalyzed modification of a chemically synthesized substance P-precursor. In the precursor peptide the residues of substance P(1-8) were flanked by tripeptide linkers on both sides. In the first step the C-terminal tripeptide amide was replaced by the authentic C-terminal tripeptide amide of substance P via alpha-chymotrypsin-catalyzed transpeptidation. The enzyme simultaneously attacks two peptide bonds in the precursor molecule leading to the formation of several side-products. The desired peptide was obtained with 25% yield. In the second step the other tripeptide linker was selectively and almost quantitatively removed from the N-terminus of the precursor via trypsin-catalyzed hydrolysis. This study demonstrates that substance P can be obtained from an engineered protein by proteinase-catalyzed processing.


Novel version of Multiple Antigenic Peptide allowing incorporation on a cysteine functionalized lysine tree. A novel version of Multiple Antigenic Peptide (MAP) is described. This approach consists of the synthesis of a properly functionalized antigen carrier and the incorporation, on request, of one or more activated antigenic peptides. This method was to synthesize a MAP containing eight epitopes of Hsp 70-1 from Plasmodium falciparum. Mice immunised with this MAP gave a specific response while those immunised with the peptide alone did not.


Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases. Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.


Simultaneous expression of keratin and glial fibrillary acidic protein by the same cells in epiretinal membranes. The identification of cells comprising epiretinal membranes is difficult because of the phenotypic changes that occur. Examination of intermediate filament protein content by immunocytochemical analysis can help to identify some cells with altered ultrastructure but is not always definitive because altered expression of intermediate filament proteins can also occur. To examine this issue further, the authors utilized a postembedding immunocytochemical technique with epiretinal membranes in which they were able to double label for keratin, a useful marker for identifying retinal pigment epithelial cells, and glial fibrillary acidic protein (GFAP), a useful marker for identifying glial cells. Nine of ten idiopathic epiretinal membranes contained cells that labeled for GFAP and not keratin. Two of these membranes also contained cells that labeled only for keratin and one membrane contained cells that simultaneously labeled for both GFAP and keratin. Other types of epiretinal membranes had an equal participation by cells that expressed only GFAP or keratin (12 of 17 membranes contained cells positive for keratin; 13 of 17 contained cells positive for GFAP). Ten of 17 nonidiopathic membranes contained cells simultaneously expressing GFAP and keratin, although they comprised only a minor subpopulation of the total number of cells present. These findings demonstrate that keratin and GFAP are not mutually exclusive intermediate filament proteins in cells of epiretinal membranes and that, although each may provide a helpful adjunct for cell type identification, neither is an absolutely specific marker.


Adhesion molecules in experimental phacoanaphylactic endophthalmitis. Intraocular accumulation of inflammatory neutrophils is an important feature of experimental phacoanaphylactic endophthalmitis (EPE). Increasing evidence suggests that localization of neutrophils to the site of inflammation requires the participation of neutrophil and endothelial adhesion molecules. These studies were undertaken to determine if blocking of adhesion molecules on neutrophils (CD18) or endothelium (ELAM-1) could attenuate EPE in Lewis rats. Treatment of experimental animals with anti-CD18 or anti-ELAM-1 significantly suppressed intraocular neutrophil accumulation, retinal hemorrhage, and vasculitis, and attenuated retinal edema formation by 48% and 70%, respectively. These observations demonstrate that antibodies directed against adhesion molecules on the neutrophil (CD18) or the vascular endothelial cell (ELAM-1) exhibit potent anti-inflammatory effects, resulting in a striking amelioration of injury in EPE in rats.


Phase II trial of fludarabine phosphate for adenocarcinoma of the pancreas. An Illinois Cancer Center study. We have conducted a phase II trial of fludarabine phosphate for advanced measurable adenocarcinoma of the pancreas. The drug was administered every 4 weeks by a daily-times-5 bolus schedule beginning at 20 mg/m2/day. No responses were observed in 20 evaluable patients, 18 of whom were previously untreated. Dose-limiting toxicity was leukopenia, and gastroenterologic side effects were frequent. Life-threatening or fatal renal dysfunction occurred in 3 patients. In this schedule fludarabine phosphate is ineffective against adenocarcinoma of the pancreas and appears to have unpredictable severe renal toxicity.


Phase II trial of fazarabine (arabinofuranosyl-5-azacytidine) in patients with advanced pancreatic adenocarcinoma. We conducted a phase II evaluation of fazarabine 1.75-2.0 mg/m2/hr over 72 hours every 28 days in 14 previously untreated patients with advanced adenocarcinoma of the pancreas. The initial dose was 1.75 mg/m2/hr in 10 patients, and 2.0 mg/m2/hr in 4 patients. The dose was escalated in 8 patients, including all 4 who started at the higher dose level. Toxicity was unexpectedly mild. The median WBC nadir was 4.4 (range: 2.4-15.8) x 10(3)/microliters, the median absolute neutrophil nadir was 3.2 (range: 0.9-13.0) x 10(3)/microliters, and the median platelet count was 134.0 (range: 48.0-291.0) x 10(3)/microliters. Gastrointestinal toxicity was generally mild. No major responses were seen, excluding, with 95% confidence, a response rate in excess of 20%.


Efficacy of prostate-specific antigen and magnetic resonance imaging in staging stage C adenocarcinoma of the prostate. The authors compared the two most common presurgical tests now used for the preoperative staging of adenocarcinoma of the prostate, prostate-specific antigen (PSA) and magnetic resonance imaging (MRI). One hundred consecutive radical retropubic prostatectomy patients were imaged at 1.5 Tesla before surgery with routine T1-weighted and T2-weighted transaxial images. The images were analyzed by two experienced radiologists for evidence of extracapsular disease. Radiologists rated each gland on a scale of 0 to 100 for the percentage likelihood of extracapsular disease based on its MRI appearance. Receiver operator characteristic (ROC) curves were plotted, and areas were calculated for the two radiologists and the preoperative PSA values. Comparison of the areas of the ROC curves generated from the two radiologists and those from the preoperative PSA values showed no statistical difference. In this series, radiologic interpretation of body coil MRI studies in those patients chosen for a radical retropubic prostatectomy was no better in staging adenocarcinoma of the prostate than simply using the preoperative PSA values.


Epitopes at the proteolytic cleavage sites of HIV-1-gp120 and RSV-F protein share a sequence homology: comparative studies with virus-induced and antipeptide antibodies. The proteolytic cleavage sites of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor gp160 and the fusion protein of respiratory syncytial virus (RSV) show a sequence homology. To study this homology two synthetic peptides corresponding to HIV-1-env-gp160-aa 507-518 (KAKRRVVQREKR) and RSV-F2-aa 130-136 (SKKRKRR) were synthesized. Human serum samples from HIV-positive or RSV-positive collections recognized the appropriate peptide in 90.6 or 37.2% respectively. No cross-reactivity towards the nonhomologous peptide could be monitored in both serum collections. In contrast, antipeptide antibodies raised against both peptides demonstrate a high degree of cross-reactivity. These data indicate that the high specificity of the virus-induced antibodies may be a result of strong conformational restrictions at the proteolytic cleavage site of both proteins. Moreover, these observations are important for diagnostic purposes. Synthetic peptides are a valuable tool for HIV antibody screening. Our data provide information concerning the specificity of antigen-antibody interaction on a highly immunogenic HIV-1 epitope.


Complement neoantigen and vitronectin are components of plaques in amyloid AL neuropathy. We studied three patients with late onset, chronic sensorimotor and autonomic neuropathy in course of plasma cell dyscrasia with Bence Jones proteinuria. Histopathological findings of nerve biopsies consisted in diffuse loss of myelinated and unmyelinated fibers associated with perivascular deposits of amorphous material with physico-chemical and ultrastructural features of amyloid. By immunohistochemistry, light chains of the same type as Bence Jones protein, components of the classic and lytic pathways of the complement and vitronectin were detected at the level of amyloid nodules. The colocalization of complement neoantigen and vitronectin suggests that this complex derives from the circulation. The elucidation of the chemical composition of amyloid might shed some light in the pathogenesis of these disorders.


[Inguinal hernia as a sequela of disordered bladder emptying?]. We examined 56 patients with hernias and 49 patients with other diseases prior to operation. They were all above 50 years of age. The urological screening examination included the patients history of the frequency of urination during night and day time, dysuria, the common risk factors (smoking, coughing, obstipation), obesity, a sonographic measurement of the prostate diameters, a sonographic evaluation of residual urine and the determination of the urinary flow rate. None of the above parameters showed a significant difference between the two groups. Surprisingly more than 60% of all men showed a pathological voiding function (either residual urine and/or a pathological flow rate). Our conclusions are: 1. Patients with inguinal hernias do not show a greater incidence of pathological bladder function than patients of the control group. The benign prostatic hypertrophy as a risk factor for the development of inguinal hernias is most questionable. 2. Because the results showed no significant difference between the two groups, a determination of residual urine prior to operation is not necessary. 3. More than 60% of the men above 50 years showed a pathological voiding function. We recommend a urological screening test for all men above 50 years of age during hospitalisation.


Molecular genetic studies in black families with sickle cell anemia and unusually high levels of fetal hemoglobin. Clinical, hematologic, and molecular genetic studies are reported for five families with SS patients having unusually high fetal hemoglobin (Hb F) levels (mean 28.3%, range 19-42%). Some of the individuals were symptom-free and one was not anemic. However, some were symptomatic despite a very high Hb F. Neither the Hb F level nor the F cell distribution entirely explained the variation in clinical severity. Molecular genetic studies identified the Senegal haplotype with the associated -158 G gamma (C----T) mutation in two of the five families. The -202 G gamma (C----G) mutation was not found in any of the individuals studied. Sequencing of the gamma-globin gene promoters to detect genetic high F determinants not detectable by restriction digestion was not performed. All AS parents and AS siblings demonstrated elevated F cells when the Senegal/-158 G gamma (C----T) mutation was present with either the beta S or beta A allele. Double heterozygosity for two different high F determinants in some SS patients is suggested by the studies in at least one family. Discordance among siblings in clinical and hematologic manifestations in two families provides additional evidence for loci regulating Hb F cell production which are not linked to the beta-globin gene clusters.


A second observation of the fetal methemoglobin variant Hb F-M-Fort Ripley or alpha 2G gamma 2(92)(F8)His----Tyr. We have identified a second baby with the fetal methemoglobin F-M-Fort Ripley. It was observed in a Caucasian infant from Canada; at least eleven additional members of that family were known to have had a neonatal cyanosis similar to that seen in the propositus and in a previously described baby (2). Sequencing of amplified DNA that included (part of) the G gamma gene greatly facilitated the characterization. The G gamma X chain was readily isolated by reversed phase high performance liquid chromatography; its quantity was approximately 12.5% of total gamma. Interestingly, the baby also carried the A gamma T mutation on one chromosome, either in cis or in trans to the G gamma X mutation. Hb F-M-Fort Ripley could be isolated in reasonably pure form by DEAE-cellulose chromatography. The isolated Hb FX was unstable, had spectral changes characteristic for the M-hemoglobins, while its methemoglobin derivative reacted rapidly with cyanide. Oxygen affinity data could not be obtained. It is suggested that the formation of a rather large amount (approximately 25%) of mixed hybrids (alpha 2G gamma X.gamma) with low oxygen affinity is the main cause for the occurrence of the neonatal cyanosis.


Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers. Gliricidia sepium and G. maculata are multi-purpose leguminous trees native to Central America and Mexico. Research programmes have been initiated to define the native distribution of Gliricidia and sample the spectrum of genetic variation. To date, there has been little systematic assessment of genetic variability in multi-purpose tree species. Accurate estimates of diversity between- and within-populations are considered a prerequisite for the optimization of sampling and breeding strategies. We have used a PCR-based polymorphic assay procedure (RAPDs) to monitor genetic variability in Gliricidia. Extensive genetic variability was detected between species and the variability was partitioned into between- and within-population components. On average, most (60 per cent) of the variation occurs between G. sepium populations but oligonucleotide primers differed in their capacity to detect variability between and within populations. Population-specific genetic markers were identified. RAPDs provide a cost-effective method for the precise and routine evaluation of variability and may be used to identify areas of maximum diversity. The approaches outlined have general applicability to a range of organisms and are discussed in relation to the exploitation of multi-purpose tree species of the tropics.


The current role of prostatic acid phosphatase and prostate-specific antigen in the management of prostate cancer. Although PSA is considered to be the true serum marker of prostatic tissue and a valuable indicator for cancer in the gland, knowledge of its significance and limitations is essential to its use for screening, staging, and monitoring CAP. PSA may be used in conjunction with DRE for early detection of CAP. Men with abnormal DRE should have a TRUS with or without biopsy. In men older than 50 years and with negative DRE and PSA < 4 ng/mL, annual evaluations are prudent. In patients with a PSA range of 4.0 to 9.9 ng/mL, high-risk groups such as black males and those with a positive family history should have TRUS. Males with negative DRE in the PSA range of 4.0 to 9.9 ng/mL should have TRUS to evaluate prostate volume and PSAD. Biopsy should be considered in those with PSAD > 0.15. Men with PSA > 10 ng/mL, even in the presence of an enlarged benign prostate, should have multiple directed biopsies under TRUS guidance.


Differential staining of glycosaminoglycans in the predentine and dentine of rat incisor using cuprolinic blue at various magnesium chloride concentrations. Rat incisors were fixed with a solution of 0.05% Cuprolinic Blue and 2.5% glutaraldehyde in the presence of various concentrations of MgCl2 according to the critical electrolyte concentration (CEC) principle. This method allows glycosaminoglycans (GAG) to be properly preserved and visualized. Small granules were stained by the cationic dye in the predentine in the absence of MgCl2. These granules grew in size and became more electron-dense when the concentration of the electrolyte was increased. Larger ribbon-like structures and granules were seen when 0.3 M MgCl2 was used. In the dentine, tiny dots in close association with the surface of the collagen fibres, or their periodic striations, were positively stained. A thick electron-dense band located on the dentine side at the predentine-dentine junction was seen both with and without 0.05 M MgCl2. With higher concentrations of the electrolyte (0.1-0.3 M), this band was reduced to a very thin line located at the border of the dentine, along with mineralizing collagen fibres. This demonstrated the presence of GAG at the dentine surface and therefore indicated that GAG may play a role as nucleator agent.


AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts. Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acid N-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.


Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.


Preparation and use of the poly-L-lysine-gold probe: a differential marker of glomerular anionic sites. The conditions required for the production of a polylysine-coated gold (PL-G) complex, which shows optimal sensitivity for the demonstration of tissue anionic sites, expressed under different conditions of pH have been investigated. Problems encountered with this complex have been compared with those found with other methods of conjugation of polylysine to colloidal gold. The performance of a bovine serum albumin (BSA)-stabilized PL-G complex was examined against other PL-G conjugates, including complexes that are commercially available, for the detection of heterogeneous glomerular anionic site populations, expressed at pH 2.5 and pH 7.0.


Immunocytochemical localization of a galanin-like peptidergic system in the brain of two urodele and two anuran species (Amphibia). Galanin-like immunoreactivity was localized in the brain of Urodela (Ambystoma, Pleurodeles) and Anura (Bufo, Xenopus) by immunocytochemistry with anti-porcine galanin antiserum. In the four species, immunoreactive perikarya were observed in the telencephalon (striatum, amygdala), diencephalon preoptic area mainly along the anterodorsal wall of the preoptic recessus, suprachiasmatic nucleus, lateral hypothalamus, ventral and dorsal infundibular nuclei, paraventricular organ, and rhombencephalon (nucleus of the solitary tract). Galaninergic fibres extended in similar regions and in the medial septum, ventral telencephalon, ventral hypothalamus, median eminence, and various mesencephalic and rhombencephalic regions. Contacts with the cerebrospinal fluid cavity occurred along the preoptic recessus (Ambystoma) and the ventral infundibular wall (all species). Fibres were scarce in the neurohypophysis. The distal and intermediate lobes of the pituitary were virtually devoid of immunoreactivity. The galaninergic system appeared more developed in adult amphibia than in young animals, suggesting the stimulating influence of sex steroids on the expression of galanin as previously described in Anguilla. The extensive distribution of the galanin-like immunoreactive neurons in amphibian brains suggests that this peptide may act as a neuromodulatur and/or neurotransmitter.


A combined ultrastructural approach to the study of nuclear matrix thermal stabilization. Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.


Cryofixed, freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens. Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (-160 degrees C) cooled by liquid nitrogen. The skin was then freeze-dried at -40 degrees C and 10(-2) atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 microns thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.


Quantitative changes of Ricinus communis agglutinin I and Helix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit. During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1 M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections using Ricinus communis agglutinin I (RCA I) or Helix pomatia lectin (HPL) to detect D-galactose- and N-acetyl-D-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per microns 2 of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p less than 0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p less than 0.01). No change was found between the initial segment and caput epididymidis. Controls showed a virtual absence of labeling. These results suggest that in addition to a multitude of changes occurring to spermatozoa during epididymal transit, there are also significant quantitative changes in the glycoconjugate content within the acrosome.


[Randomized double-blind study of therapy of sudden deafness. Low molecular weight dextran + naftidrofuryl vs. low molecular weight dextran + placebo]. Eighty patients with idiopathic sudden deafness existing no longer than 10 days were included in a prospective randomized double-blind study. Patients were treated for 10 days with infusions of either 10% low-molecular weight dextran or the combination of low-molecular weight dextran with naftidrofuryl. Before treatment and after 10 days hearing loss in the affected ear was determined at 0.5, 1, 2, 3, 4 and 6 kHz. The mean hearing loss was then calculated as the average from these values. During monotherapy with low-molecular weight dextran the mean hearing loss decreased from 40 to 27 dB compared to 38 to 17 dB when naftidrofuryl treatment was added (p < 0.01 between groups). A significant benefit of naftidrofuryl on hearing loss was also found at frequencies between 0.5 and 3 kHz. Furthermore, patients reported better improvement of tinnitus when naftidrofuryl was combined with dextran. Two patients receiving dextran alone developed side effects: one had an allergic reaction causing withdrawal of treatment, which the other case had vertigo, nausea and headache with spontaneous recovery. The results of the study showed that treatment with naftidrofuryl in addition to hemodilution with low-molecular weight dextran was of therapeutic benefit in the therapy of sudden deafness without increasing the rate of side effects.


Mapping of an epitope defined by a human hybridoma antibody (TrD3): a new HLA-B supertype associated with a subset of HLA-Bw6. The new human-human hybridoma TrD3 secretes a cytotoxic IgM mAb, which reacted with 28 of a panel of 56 HLA-typed lymphoblastoid cells. All 28 TrD3+ cells expressed the HLA-B supertype Bw6, whereas 10 Bw6+ cells were not recognized by the mAb. None of the 17 Bw4 homozygous cells were positive with TrD3. Thus, TrD3 divided the Bw6+ HLA-B specificities of the cell lines into two subgroups, namely, Bw6+TrD3+ and Bw6+TrD3-, and therefore defines a new HLA-B supertype. TrD3 reacted strongly with some B8+ cell lines and weakly or not at all with others, suggesting a new split of HLA-B8. Compared with cell lines, TrD3 reacted more weakly with freshly isolated T cells from blood. The Bw6-specific rat mAb SFR8-B6 partially blocked the binding of 125I-labeled TrD3 to a Bw6+ cell line. By using cell lines transfected with hybrid genes between HLA-B7 (Bw6+) and HLA-B27 (Bw6-) as targets in flow cytometry, critical residues for the TrD3 epitope could be mapped to the amino acid region 24-62 of the HLA class-I alpha 1 domain. Comparison of deduced amino acid sequences of TrD3-positive and -negative cells indicated that a tryptophane residue at position 95 destroyed the TrD3 epitope, and that one or more of the residues in positions 24, 45, and 46 may be critical, suggesting that it is a discontinuous epitope. It is notable that none of these residues are located on alpha-helixes.(ABSTRACT TRUNCATED AT 250 WORDS)


Signal transduction through crosslinking CD7 and IgM-Fc receptors that inhibits T-cell proliferation. We have previously suggested a possible involvement of CD7 and/or the putative receptor for IgM-Fc (FcRmu) in the downregulation of T-cell responses to mitogen and alloantigen. In this report, we examined the effect of ligand-receptor interaction upon T-cell proliferation by using anti-CD7 monoclonal antibodies (mAbs) and purified human IgM (HIgM) and showed that crosslinking CD7 and/or FcRmu on T cells resulted in significant inhibition of mitogen- and alloantigen-induced proliferative responses. In most cases, crosslinking of FcRmu alone or together with CD7 receptors on peripheral blood lymphocytes (PBLs) resulted in more potent suppression of T-cell proliferation than that caused by crosslinking CD7 alone. Examination of potential inhibitory mechanisms revealed that crosslinking CD7 and FcRmu does not reduce cell viability or the expression of T-cell markers that are important for T-cell activation or proliferation. However, crosslinking CD7 and/or FcRmu of PBLs significantly reduced phytohemagglutinin (PHA)-induced IL-2 production and rendered PBLs unable to utilize exogenously added human recombinant IL-2 (rIL-2). Further examination of possible signal transduction that might be associated with crosslinking CD7 and/or FcRmu receptors indicated a marked reduction in the magnitude and duration of intracellular calcium (Ca++)i released in response to PHA. These findings suggest that crosslinking CD7 and FcRmu inhibits T-cell activation and proliferation by a calcium-dependent mechanism that inhibits IL-2 production and/or utilization.


Developmentally-regulated coexpression of vimentin and cytokeratins in the rat inner ear. In the present study the expression of vimentin-type intermediate filament proteins and cytokeratins was studied immunohistochemically in the rat inner ear from 12 days postconception up to 40 days after birth. With the use of a broad spectrum monoclonal antibody, cytokeratin expression was found to be present in the whole epithelial lining except for the sensory cells, throughout all the developmental stages examined. Vimentin was detected in the mesenchymal cells, the mesenchyme-derived tissues and the intermediate cells of the stria vascularis, confirming their origin from melanocyte precursor cells. In addition, the coexpression of vimentin and cytokeratins in the epithelial lining of the membranous inner ear was found to be developmentally regulated. During the final stages of differentiation, vimentin expression disappeared from the majority of the cell types. In the mature cochlea the coexpression of vimentin and cytokeratins was still found in the supporting cells of the organ of Corti, in the cells of Claudius and in external sulcus cells. As far as we could conclude from this study, the sensory cells showed only vimentin expression but not cytokeratin expression. A possible relationship between vimentin expression in adult epithelial cells of the inner ear and a specialised function of these cells is discussed.


Connections between the cochlear nuclei in guinea pig. This study provides a detailed analysis of the appearances and distributions of neurons projecting from one cochlear nucleus to the other. Injections of wheatgerm agglutinin conjugated to horseradish peroxidase were made into ventral or dorsal cochlear nucleus of the guinea pig. Retrogradely labeled cells in the opposite cochlear nucleus were examined and quantified. Three major categories of labeled cells were discerned on the basis of their soma shape: elongate, round-to-oval, and polygonal. All injections resulted in widespread labeling of cells in all of these categories, but especially round-to-oval cells, in the opposite ventral cochlear nucleus and sparse labeling in the dorsal cochlear nucleus. The results suggest that there is a significant cochlear nucleus commissural projection involving heterogeneous cell types which could have diverse functions in binaural auditory signal processing.


Characterization of marginal and Claudius' cells growing from cochlear explants in vitro. Tissue specimens of stria vascularis together with spiral ligament were transferred from the guinea pig cochlea to tissue culture dishes. To characterize and identify cells growing out from the explants, indirect immunofluorescence microscopy was used. The expression of the intermediate-sized filaments vimentin and cytokeratin 18 in cells on the surface of tissue specimens and in cells growing out from the explants after different cultivation periods were compared. Basically, three types of cells grew from the explants during several days: marginal cells, Claudius' cells and fibroblast-like cells. In primary cultures of explants, growth of marginal cells was observed in 25% of the dishes. Their proliferative activity, estimated by the use of the BrdUrd-DNA antibody, started in the stria vascularis and continued across the attachment of Reissner's membrane down to the bottom of the cell culture dish. The newly-formed marginal cells expressed cytokeratin 18 in the same way that original marginal cells on the tissue specimen do. If the newly-formed marginal cells were in contact with fibroblast-like cells or were forming groups (domes) on the bottom, they expressed vimentin. In 3% of the dishes growth of Claudius' cells was observed. Proliferative activity of these cells was found at the point where the basilar membrane was attached to the spiral ligament. New Claudius' cells spread at the opposite side of an explant when compared with the location of new marginal cells. Original as well as newly-formed Claudius' cells contained cytokeratin 18. Fibroblast-like cells were commonly present in cultures and contained only vimentin.


Effect of porcine somatotropin on number of granulosa cell luteinizing hormone/human chorionic gonadotropin receptors, oocyte viability, and concentrations of steroids and insulin-like growth factors I and II in follicular fluid of lean and obese gilts. Prepubertal gilts of obese (n = 24) or lean (n = 24) genetic lines were injected (s.c.) daily with 0, 2, or 4 mg of porcine somatotropin (pST) for 6 wk starting at 160 d of age to determine whether pST affects follicular function. Blood and ovaries were collected at slaughter 24 h after the last injection. Surface follicles greater than or equal to 1.0 mm in diameter were counted, and pools of follicular fluid (FFL) and granulosa cells were collected from 1.0- to 3.9-mm (small) and 4.0- to 6.9-mm (medium) follicles. Oocytes were collected from small and medium follicles and evaluated for maturational stage and viability. Porcine somatotropin increased (P less than .08) the numbers of small but not the numbers of medium follicles per gilt (P greater than .10). Oocyte maturation and viability were not affected by pST or genetic line. Porcine somatotropin increased (P less than .05) concentrations of insulin-like growth factor I (IGF-I) in serum and FFL of both obese and lean gilts; IGF-I was lower (P less than .01) in lean gilts. Treatment with pST decreased (P less than .05) IGF-II in FFL of lean but not in that of obese gilts. Dose of pST and line had no effect on concentrations of progesterone in FFL of small or medium follicles or on concentrations of estradiol in FFL of small follicles.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparison of the lethal effects in culture of cytosine arabinoside and arabinofuranosyl-5-azacytosine acting on the blast cells fo acute myeloblastic leukemia. Two culture methods are available for the study of the blast cells of acute myeloblastic leukemia (AML); first, a minority of blast cells will form colonies in methylcellulose cultures in the presence of suitable growth factors. Second, clonogenic blast cells will increase in suspension cultures. Both methods can be used to assess the sensitivity of blasts to chemotherapeutic drugs, but different dose-response curves are obtained with each. Thus cytosine arabinoside (ara-C) is more toxic to clonogenic blasts in suspension than in methylcellulose, while for 5-azacytidine (5-aza) the reverse is seen. Arabinofuranosyl-5-Azacytosine (ara-AC) combines the chemical features of the two drugs. That is, its sugar moiety has the same diastereomeric change in the furanose ring as ara-C and its pyrimidine ring has the same substitution of nitrogen for carbon at the 5' position as 5-aza. We compared the sensitivity of AML blasts with ara-Ac in suspension and in methylcellulose. As a control, the same comparison was made for the sensitivities to ara-C. Blast cells were less sensitive to ara-AC than to ara-C under all conditions; but, ara-AC sensitivity was greater for cells in methylcellulose than for cells in suspension. Thus, AML blasts respond to ara-AC in culture with a pattern similar to that of 5-aza and opposite to that of ara-C. Since ara-C is a more useful drug in the treatment of AML than 5-aza, we interpret the culture results as predicting that ara-AC may not be effective in AML.


Effects of serum acid phosphatase [correction of phosphate] elevation following transurethral prostatectomy on long-term mortality. Studies of surgery for symptoms of bladder outlet obstruction in men suggest that a possible higher long-term mortality occurs in patients having transurethral prostatectomy compared with patients having an open prostatectomy. It is the purpose of this study to determine if intraoperative factors affect the long-term survival of patients having transurethral prostate resection for benign prostate hypertrophy. In 158 consecutive patients having transurethral prostatectomy for benign adenoma who were followed for eight years, 28 patients died during the follow-up period. In comparing those patients who are alive with those patients who have died, there was no significant difference at the time of surgery in intraoperative irrigant absorption as indicated by changes in serum sodium and there was no significant difference in the intraoperative absorption of prostate tissue substances as indicated by changes in serum acid phosphatase. The only factor in this study associated with long-term survival was age of the patient at the time of surgery with older patients having a higher long-term mortality. This study suggests that age of the patient rather than intraoperative factors is associated with long-term survival following transurethral prostatectomy.


Physical map of the Listeria monocytogenes chromosome. The circular physical map of the pathogenic bacterium Listeria monocytogenes LO28 (serovar 1/2c) was established by using pulsed-field gel electrophoresis. The L. monocytogenes chromosome contains eight NotI fragments of 1,100, 940, 400, 335, 280, 45, 30, and 20 kb in size and eight Sse8387I fragments of 860, 680, 680, 370, 335, 130, 70, and 25 kb. Therefore, the total length of the genome is 3,150 kb. To order the NotI fragments on the chromosome, we used a strategy which can be of general use. We first cloned chromosomal HindIII or EcoRI fragments in pBR322. DNA extracted from the total libraries was digested by NotI and ligated to a NotI-kanamycin resistance cassette obtained by cutting Tn5 with NotI. After transformation in Escherichia coli, kanamycin-resistant clones originating from NotI-containing EcoRI or HindIII fragments were isolated. The two EcoRI-NotI or HindIII-NotI fragments of each recombinant plasmid were isolated and used as probes on Southern blot hybridizations to identify and link the corresponding NotI fragments. Seven NotI fragments were ordered in this way. The last junction was demonstrated by partial digest analysis. All L. monocytogenes genes identified so far as well as the six rRNA operons were localized on the NotI map. Regions homologous to genes from closely related bacteria were also detected and localized. Southern blot analysis of simple Sse8387I digests or double Sse8387I-NotI digests probed with the various NotI probes allowed us to align the Sse8387I fragments and localize the single SfiI site, resulting in the establishment of the first genetic and physical map of the L. monocytogenes chromosome.


Scleral fibroblasts of the chick embryo differentiate into chondrocytes in soft-agar culture. Scleral fibroblasts, perichondrial cells of the scleral layer of the 12-day chick embryo, always manifest a fibroblastic morphology in monolayer culture. In soft-agar culture, these cells produce two types of colonies. One type of colony, F-type, consists of adherent fibroblastic cells, and the other, C-type, is composed of scattered round chondrocytic cells. Cells of the C-type colony are surrounded by a halo of extracellular matrix, positive with Alcian blue and with an antibody to cartilage-specific proteoglycan. When a single fibroblast clone in monolayer, derived from a single scleral fibroblast, is subcultured into soft agar, the cells give rise to both C-type and F-type colonies. Further, it was found that cells constituting F-type colonies eventually separate and become spherical, and the F-type colony converts to a C-type colony (C-type conversion). In regard to the C-type convertibility, the primary fibroblast clones were divided into four categories, early time differentiating, middle-time differentiating, late-time differentiating and nondifferentiating. This suggests that the scleral perichondrial layer of the 12-day chick embryo is composed of a variety of cells with different chondrogenic potentialities maintained in each individual cell.


How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32. In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.


Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D. Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834, 1991) was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdhA and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH II proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bdhA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations.


Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes. The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.


Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen. The rfp gene of Shigella dysenteriae 1 and the rfa genes of Escherichia coli K-12 and Salmonella typhimurium LT2 have been studied to determine their relationship to lipopolysaccharide (LPS) core heterogeneity and their role in the attachment of O antigen to LPS. It has been inferred from the nucleotide sequence that the rfp gene encodes a protein of 41,864 Da which has a structure similar to that of RfaG protein. Expression of this gene in E. coli K-12 results in the loss of one of the three bands seen in gel analysis of the LPS and in the appearance of a new, more slowly migrating band. This is consistent with the hypothesis that Rfp is a sugar transferase which modifies a subset of core molecules so that they become substrates for attachment of S. dysenteriae O antigen. A shift in gel migration of the bands carrying S. dysenteriae O antigen and disappearance of the Rfp-modified band in strains producing O antigen suggest that the core may be trimmed or modified further before attachment of O antigen. Mutation of rfaL results in a loss of the rough LPS band which appears to be modified by Rfp and prevents the appearance of the Rfp-modified band. Thus, RfaL protein is involved in core modification and is more than just a component of the O-antigen ligase. The products of rfaK and rfaQ also appear to be involved in modification of the core prior to attachment of O antigen, and the sites of rfaK modification are different in E. coli K-12 and S. typhimurium. In contrast, mutations in rfaS and rfaZ result in changes in the LPS core but do not affect the attachment of O antigen. We propose that these genes are involved in an alternative pathway for the synthesis of rough LPS species which are similar to lipooligosaccharides of other species and which are not substrates for O-antigen attachment. All of these studies indicate that the apparent heterogeneity of E. coli K-12 LPS observed on gels is not an artifact but instead a reflection of functional differences among LPS species.


The luxR gene product of Vibrio harveyi is a transcriptional activator of the lux promoter. Expression of the lux operon from the marine bacterium Vibrio harveyi is dependent on cell density and requires an unlinked regulatory gene, luxR, and other cofactors for autoregulation. Escherichia coli transformed with the lux operon emits very low levels of light, and this deficiency can be partially alleviated by coexpression of luxR in trans. The V. harveyi lux promoter was analyzed in vivo by primer extension mapping to examine the function of luxR. RNA isolated from E. coli transformed with the Vibrio harveyi lux operon was shown to have a start site at 123 bp upstream of the first ATG codon of luxC. This is in sharp contrast to the start site found for lux RNA isolated from V. harveyi, at 26 bp upstream of the luxC initiation codon. However, when E. coli was cotransformed with both the lux operon and luxR, the start site of the lux mRNA shifted from -123 to -26. Furthermore, expression of the luxR gene caused a 350-fold increase in lux mRNA levels. The results suggest that LuxR of V. harveyi is a transcriptional activator stimulating initiation at the -26 lux promoter.


Aggression, suicidality, and serotonin.

Aggression, suicidality, and serotonin. Studies from several countries, representing diverse cultures, have reported an association between violent suicide attempts by patients with unipolar depression and personality disorders and low concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid (CSF). Related investigations have documented a similar inverse correlation between impulsive, externally directed aggressive behavior and CSF 5-HIAA in a subgroup of violent offenders. In these individuals, low CSF 5-HIAA concentrations are also associated with a predisposition to mild hypoglycemia, a history of early-onset alcohol and substance abuse, a family history of type II alcoholism, and disturbances in diurnal activity rhythm. These data are discussed in the context of a proposed model for the pathophysiology of a postulated "low serotonin syndrome."


Catalytic properties of yeast protein kinase C: difference between the yeast and mammalian enzymes. With bovine myelin basic protein as a model common substrate, protein kinases C (PKC) purified from yeast (Saccharomyces cerevisiae) and mammalian tissue (rat brain) were shown to exhibit clearly different catalytic properties. The major sites of phosphorylation in bovine myelin basic protein by the yeast PKC were identified: Thr-19, Thr-34, and Thr-65. These sites are distinctly different from those for the mammalian PKC: Ser-8, Ser-46, Ser-55, Ser-110, Ser-132, Ser-151, and Ser-161, which were previously identified [Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., & Nishizuka, Y. (1985) J. Biol. Chem. 160, 12492-12499]. The results suggest that the yeast and mammalian enzymes may play distinct roles in cellular regulation. No evidence is available, however, that a yeast-type PKC exists in mammalian tissues. An oligopeptide containing the sequence around Thr-19 of bovine myelin basic protein, Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr(19)-Met-Asp-His-Ala, can be used as a substrate for selective assaying of the yeast PKC.


Inhibitory effects of triphosphate derivatives of oxetanocin G and related compounds on eukaryotic and viral DNA polymerases and human immunodeficiency virus reverse transcriptase. In order to clarify the biological activities of (-)-oxetanocin G, and (-)-oxetanocin A and its carbocyclic analogue, (-)-carboxetanocin G, the inhibitory effects of triphosphate derivatives of these compounds (OXT-GTP, OXT-ATP, and C-OXT-GTP) on eukaryotic and viral DNA polymerases were examined. DNA polymerase alpha purified from calf thymus was weakly inhibited by OXT-GTP and OXT-ATP but strongly by C-OXT-GTP, the Ki value being 0.22 microM. On the other hand, rat DNA polymerase beta was not affected by these analogues. DNA polymerase gamma purified from bovine testes was very weakly inhibited by OXT-GTP and OXT-ATP, but not by C-OXT-GTP. DNA polymerase from herpes simplex virus type-II (HSV-II) was strongly inhibited by all three analogues, the Ki values ranging from 0.5 to 1.0 microM. Human immunodeficiency virus-encoded reverse transcriptase (HIV RT) was also strongly inhibited by these three analogues, the Ki value of C-OXT-GTP being slightly smaller than that of OXT-GTP or OXT-ATP. Analysis of products synthesized on singly primed M13 single-stranded DNA by DNA polymerase alpha, HSV-II DNA polymerase or HIV RT in the presence of the analogues revealed that OXT-GTP and C-OXT-GTP were incorporated into DNA and caused chain termination mainly at sites one or two nucleotides beyond the cytosine bases on the template.


The reversible and irreversible autophosphorylations of insulin receptor kinase. When the catalytically active, tyrosyl-phosphorylated form of insulin receptor was isolated from human placenta and treated with ADP, only partial dephosphorylation was observed. This observation suggests the existence of two distinct classes of phosphotyrosyl residues of the phosphorylated insulin receptor: one in which the phosphoryl groups undergo reversible transfer to ADP and one in which they do not. There were 8.8 +/- 2.2 phosphorylation sites per tetrameric insulin receptor, of which 2.4 +/- 0.5 were irreversible (mean +/- S.D., n = 6). The reversible sites were determined to be equivalent and noninteracting and to have an equilibrium constant for the transfer of a phosphoryl group from ATP to a site of reversible phosphorylation of 8.7. Surprisingly, both phosphorylation and dephosphorylation at the reversible sites were relatively insensitive to the presence of insulin. Since only minimal autophosphorylation of insulin receptor was detected in the absence of insulin, the results suggest that the incorporation of phosphate into the two to three irreversible phosphorylation sites is insulin dependent. Phosphorylation of the irreversible phosphorylation sites then renders the insulin receptor relatively insensitive to the continued presence of insulin and facilitates rapid reversible phosphorylation of a second group of tyrosyl residues. The dependence of the degree of phosphorylation of insulin receptor on the ATP:ADP ratio may provide a mechanism for modulating the cellular response to insulin.


Catalytic properties of the cloned amylase from Bacillus licheniformis. A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.


Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes. The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.


Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat. Growth factors stimulate the enzyme phosphatidylinositol (PI) 3-kinase in cells in culture. Insulin rapidly stimulates tyrosine phosphorylation of its endogenous substrate, insulin receptor substrate 1 (IRS-1), and in vitro IRS-1 associates with PI 3-kinase, thus activating the enzyme. We have examined whether insulin is capable of stimulating the PI 3-kinase pathway in two physiological target tissues for the actions of insulin in vivo, liver and skeletal muscle. After intraportal injection of insulin into anesthetized rats, there was a 2-fold stimulation of total hepatic PI 3-kinase activity in liver and muscle extracts and a 10- to 20-fold increase in PI 3-kinase activity immunoprecipitated with anti-IRS-1 antibodies. Stimulation of PI 3-kinase was accompanied by an association between this enzyme and IRS-1 as detected by immunoprecipitation of liver and muscle extracts with anti-IRS-1 antibodies and Western blotting with antibodies to the 85-kDa subunit of PI 3-kinase. Immunoprecipitation with anti-p85 antibodies and phosphotyrosine immunoblotting revealed no tyrosine phosphorylation of PI 3-kinase, but demonstrated co-precipitation of tyrosine-phosphorylated IRS-1, as well as another phosphotyrosine protein of approximately 135-140 kDa. Thus, IRS-1 phosphorylation plays a significant role in the activation of PI 3-kinase in vivo by insulin.


Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.


A T-cell enhancer cooperates with NF-kappa B to yield cytokine induction of E-selectin gene transcription in endothelial cells. ELAM1 (endothelial leukocyte adhesion molecule 1, also known as E-selectin) is a highly tissue-specific adhesion molecule that is transiently and exclusively expressed on cytokine-induced endothelial cells. We have identified two proximal ELAM1 promoter elements and their DNA-binding factors that are, in addition to NF-kappa B, essential for ELAM1 transcription. Mutation of either element in promoter constructs carrying the first 383 nucleotides of the ELAM1 promoter markedly diminshed the expression of a fused chloramphenicol acetyltransferase reporter gene. Although multimers of either element failed to display enhancer activity on its own, fusion of the most upstream of these to the NF-kappa B element had a strong stimulatory effect. This site, ACATCAT, is recognized by a factor we have called NF-ELAM1. The site corresponds to NF-ELAM1's preferential binding sequence (A/T)CA(G/T)CA(G/T) as determined in a target definition assay. This element is identical to the T-cell delta A enhancer found in the T-cell receptor-alpha, -beta, and CD3 delta genes. Our results suggest that the delta A/NF-ELAM1 element can function as a modulator of NF-kappa B in endothelial cells both as well as a T-cell enhancer.


N-glycosylation is required for human CD2 immunoadhesion functions. The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.


Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action. Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.


Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo. After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.


N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness. To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.


Expression and function of IRS-1 in insulin signal transmission. IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.