Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks.

We have recently shown that ilimaquinone (IQ) causes the breakdown of Golgi membranes into small vesicles (VGMs for vesiculated Golgi membranes) and inhibits vesicular protein transport between successive Golgi cisternae (Takizawa et al., 1993). While other intracellular organelles, intermediate filaments, and actin filaments are not affected, we have found that cytoplasmic microtubules are depolymerized by IQ treatment of NRK cells. We provide evidence that IQ breaks down Golgi membranes regardless of the state of cytoplasmic microtubules. This is evident from our findings that Golgi membranes break down with IQ treatment in the presence of taxol stabilized microtubules. Moreover, in cells where the microtubules are first depolymerized by microtubule disrupting agents which cause the Golgi stacks to separate from one another and scatter throughout the cytoplasm, treatment with IQ causes further breakdown of these Golgi stacks into VGMs. Thus, IQ breaks down Golgi membranes independently of its effect on cytoplasmic microtubules. Upon removal of IQ from NRK cells, both microtubules and Golgi membranes reassemble. The reassembly of Golgi membranes, however, takes place in two sequential steps: the first is a microtubule independent process in which the VGMs fuse together to form stacks of Golgi cisternae. This step is followed by a microtubule-dependent process by which the Golgi stacks are carried to their perinuclear location in the cell. In addition, we have found that IQ has no effect on the structural organization of Golgi membranes at 16 degrees C. However, VGMs generated by IQ are capable of fusing and assembling into stacks of Golgi cisternae at 16 degrees C. This is in contrast to the cells recovering from BFA treatment where, after removal of BFA at 16 degrees C, resident Golgi enzymes fail to exit the ER, a process presumed to require the formation of vesicles. We propose that at 16 degrees C there may be general inhibition in the process of vesicle formation, whereas the process of vesicle fusion is not affected.

Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65.

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.

Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators.

The 2 micron plasmid of Saccharomyces cerevisiae is maintained by the action of plasmid-encoded gene products that control copy number and promote equipartition of plasmid copies at cell division. We show that the REP1 and REP2 plasmid-encoded gene products are master regulators that act in concert to autoregulate the level of their own transcripts and to regulate transcript levels of the FLP gene that promotes plasmid copy amplification. REP1 and REP2 are also shown to repress transcription at REP3, the cis-acting site essential for plasmid equipartitioning. We propose a model in which REP3 acts by dislodging transcription apparatuses that otherwise cause plasmid molecules to adhere to the mother nucleus and segregate asymmetrically. On the basis of their ability to generate specific chromatin structures, we also propose that the REP1 and REP2 gene products interact with different specific sequences found iterated in the 2 micron plasmid.

Identification of a leukemia-associated antigen of human acute lymphocytic leukemia.

A human leukemia-associated antigen (LAA) has been identified by immunofluorescence and electrophoretic analyses. LAA was detected on the surfaces of cells from patients with acute lymphocytic leukemia (ALL) as well as on the surfaces of leukemia cells from the established cell lines NALM-1, NALM-16, MOLT-4, CCRF-CEM, and RPMI 8402. The antigen was not detected on BALM-1 or Raji cells (established B-cell lines), bone marrow cells from ALL patients in remission, or on blood lymphocytes from normal donors. This antigen was most frequently associated with common ALL (cALL); however, cells from 2 of 12 patients with T-cell ALL and 1 patient with B-cell ALL also expressed this antigen. Under reduced conditions, the antigen had an approximate molecular mass of 100,000 daltons as determined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiographic analysis and appeared to be the same cALL antigen that has recently been described by others. The probability that LAA is a normal differentiation antigen was discussed.

Increased adenosine deaminase (ADA) activity and a shift from ADA-dependent to ADA-independent phases during T-cell activation: a paradox.

During activation of WF rat splenic T-cells, a change occurs with respect to susceptibility to a toxic accumulation of adenosine or deoxyadenosine (dADO) in the presence of adenosine deaminase (ADA) blockade. Addition of nucleoside 1 hour after the initiation of a concanavalin A response in the presence of 2'deoxycoformycin (DCF) markedly inhibited the response, whereas delay of addition of the nucleoside for 24-48 hours resulted in minimal or no inhibition. Inhibition was not simply the result of prolonged incubation of cells in the presence of nucleoside and was apparently not attributable to an effect on proliferating cells. Addition of interleukin 2 (IL-2) to cultures containing DCF and dADO did not reverse the inhibitory effect, which suggests that IL-2-producing T-cells also were not the target of nucleoside toxicity. A twofold increase in ADA activity that occurred during T-cell activation was nonessential for the survival of mitogen-activated T-cells in the presence of toxic concentrations of dADO and did not account for an apparent increased resistance of these cells to nucleoside toxicity. These paradoxical observations prompted an analysis of ADA activity in various populations of activated T-cells enriched with cells in G0/G1, S, or G2+M cell-cycle phases, which indicated that increased ADA activity was not associated with a specific period during cell-cycle traverse, but, rather, coincided with cell enlargement in preparation for mitosis. In conclusion, either an early event in T-cell mitogenesis is highly susceptible to nucleoside toxicity or a mechanism independent of ADA is acquired during T-cell activation that allows proliferating T-cells to resist toxic concentrations of nucleoside.

A tandem duplication causes the Kn1-O allele of Knotted, a dominant morphological mutant of maize.

Molecular and genetic techniques are used to define Kn1-O, a mutation which interferes with the normal differentiation of vascular tissue in leaves. Sequences associated with a previously cloned allele, Kn1-2F11, were used as hybridization probes in a Southern analysis of Kn1-O. By this analysis, Kn1-O lacks the Ds2 transposable element that causes Kn1-2F11 but instead is associated with a sequence duplication. Sequence and restriction analysis of genomic clones show that the duplication consists of a tandem array of two 17-kb repeats. Analysis of Kn1-O derivatives indicates that the duplication itself conditions the mutant phenotype; a severely knotted line, Kn1-Ox, has gained a repeat unit to form a triplication, whereas normal derivatives have either lost a repeat unit or sustained insertions that disrupt the tandem duplication. These insertions map near the central junction of the tandem duplication, suggesting that the mutant phenotype results from the novel juxtaposition of sequences. We discuss models that relate the tandem duplication of sequences to altered gene expression.

Measles revaccination response in a school-age population.

Due to the dramatic upsurge in the incidence of measles, the American Academy of Pediatrics and the Immunization Practices Advisory Committee of the Centers for Disease Control revised their measles immunization policies in 1989 to include a routine two-dose schedule. The objectives of this study were the following: (1) determine the prevalence of immunologically measles-susceptible subjects in a previously vaccinated, school-age, military dependent population; and (2) assess risk factors to identify immunologically measles-susceptible subjects. Serum was collected just prior to measles revaccination and again 2 weeks later. Measles-specific IgG and IgM titers were determined by enzyme-linked immunosorbent assay. Immunologically measles-susceptible subjects constituted 9.8% of the population. The interval since previous measles vaccination was significantly related to pre- and postrevaccination IgG titers in a repeated-measures analysis of variance model. The magnitude of increase in IgG titer following revaccination and analysis of trend for proportions of measles-susceptible subjects were significantly related to the age of initial vaccination. This study supports continued measles revaccination; in addition, revaccination appears to be of greater value at 11 to 12 years of age than at 4 to 6 years of age.

Identification and characterization of a human thymus-leukemia antigen (43-kDa/513TL) bearing a structural relationship to HLA.

A human thymus-leukemia-like antigen has been identified that is antigenically distinct from T6/HTA-1. This was accomplished with a rabbit antiserum (513) which was prepared against lymphoblasts that were E rosette negative (E-), human thymus antigen positive (HuTA+), cALLA-, DR-, SmIg- from a patient who presented with a mediastinal mass and a WBC count of 130 X 10(9) cells/1. Following absorption with B cell and "null" cell lines, 513 exhibited prominent reactivity with a membrane antigen that was present on normal thymocytes and lymphoblasts from 11 of 13 patients with T cell ALL and 1 of 16 patients with common ALL, but was not detected on normal peripheral blood lymphocytes, normal bone marrow cells and leukemic lymphoblasts with an undifferentiated phenotype. SDS-PAGE analysis of this antigen indicated that it was composed of two subunits, 43-kDa and 12-kDa. Sequential absorption experiments revealed that: (1) the 12-kDa subunit was antigenically similar to beta 2 microglobulin; (2) the intact molecule did not exhibit HLA-A, B or C "framework" determinants; (3) the molecule was antigenically distinct from a human thymus-leukemia antigen HTA-1 (recognized by monoclonal antibodies NA1/34 and OKT6); and (4) the molecule was antigenically distinct from adenosine deaminase. It is concluded that 513 reacts with a membrane protein (designated 513TL) which exhibits properties characteristic of a histocompatibility-like antigen whose expression is restricted to thymocytes and some leukemias (TL antigen). Its antigenic distinction from another recently characterized human TL antigen, HTA-1, suggests polymorphism among this family of alloantigens.

Neutral and acidic human tracheobronchial mucin. Isolation and characterization of core protein.

Human bronchial mucin from a patient suffering from chronic bronchitis was solubilized in aqueous solution containing sodium azide and protease inhibitors and purified by Sepharose 4B and 2B column chromatography. The mucin was further purified by cesium bromide density gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis of this material showed high-molecular-weight mucin component(s) at the top of the gel. Chemical analysis of this preparation indicated a typical mucin profile of amino acids and carbohydrates. Ion-exchange chromatography resulted in resolution of the purified mucin into neutral and acidic fractions. Comparison of the chemical composition of these two fractions showed higher mole percentage of threonine, serine, sialic acid, and sulfate in the acidic fraction. Chemical deglycosylation of the purified mucin preparation with trifluoromethane sulfonic acid was carried out at 20 degrees C for 3 1/2 h. Sialic acid, fucose, galactose, and N-acetylglucosamine were completely removed, whereas traces of N-acetylgalactosamine were still detected. High-pressure liquid chromatography of the deglycosylated products from native, neutral, and acidic mucin preparations resulted in a principal peptide, P1, with identical amino acid composition. Cyanogen bromide (CNBr) treatment of the peptide P1 from neutral and acidic mucins and subsequent fractionation of the fragments by high-pressure liquid chromatography resulted in similar peptide profiles. The P1 peptide fraction was further subjected to high-pressure liquid chromatography in a second solvent system, which resulted in two peaks, P1a and P1b. Gel filtration of both peptides in 6 M guanidine hydrochloride indicated a single peak with molecular weight of approximately 97 kDa. The amino acid profile of the two peptides was dominated by high levels of threonine, serine, and proline, which combined accounted for nearly 39% of the total residues, and in most respects, the profile resembled that of native mucin. End-group analysis of the peptide P1a indicated a blocked N-terminus, whereas serine was found to be the N-terminal amino acid in the peptide P1b. Rabbit antibodies prepared against the peptide P1 from native tracheal mucin reacted strongly with neutral and acidic mucin as well as the mucin from human colon. Both neutral and acidic human tracheal mucins were immunologically reactive with mouse monoclonal antibody HMPFG-2, which was prepared against human mammary mucin. However, the response of this antibody to human colonic mucin was rather weak.

Serological response to measles revaccination in a highly immunized military dependent adolescent population.

In the spring of 1986, there was a measles outbreak in the city of El Paso, Texas, with 92 cases reported to the City-County Health Department. Of those 92 cases, 31 (32%) occurred within a public high school's student population of 2524. A mass measles vaccination program was undertaken at that high school in order to limit the outbreak. The student enrollment included a military dependent population of 368 students. Despite documented histories of prior measles immunizations in this military dependent subgroup, three individuals contracted the disease. Since this subgroup of students represented a highly immunized adolescent population, it was of interest to serologically determine their immune status prior to and following reimmunization with the expectation that such a study would provide information relating to the level of "protective" immunity. Prevaccination and postvaccination sera were obtained from 95 students. Results of measuring anti-measles antibody activity by ELISA indicate that 13 (14%) students responded to revaccination and experienced a fourfold or greater rise in IgG antibody levels. There were no detectable IgM responses. All of the students who responded to revaccination produced an anamnestic response (IgG boost only). Since most of these individuals had received first immunizations at 15 months of age or older, these findings suggest that secondary vaccine failure (waning immunity) was responsible for the putative "lowered" immunity in these individuals, instead of primary vaccine failure (maternal antibody suppression). These findings support current recommendations for measles booster revaccination of school-age children and adolescents.

To reuse your circuit: the HME debate.

Heat and moisture exchangers (HMEs) have been used for more than 30 years for heat and moisture retention during general anesthesia. Studies about bacteriostatic vs nonbacteriostatic HMEs (BHMEs/NHMEs) have been conducted to assess their role in preventing bacterial transmission to the anesthesia breathing circuit; none have been done on anesthetized patients in the operating room. The present study adds to existing knowledge about the HME's ability to prevent transmission of bacteria, with implications for cost reduction resulting from reuse of anesthesia breathing circuits among patients. The chi 2 test revealed no statistically significant differences between groups in transmission of bacteria from endotracheal tube (ETT) to anesthesia breathing circuit (P = .48). However, both groups showed statistically significant differences between presence of bacteria in ETTs and anesthesia breathing circuits: Group 1, BHME (P < .005) and group 2, NHME (P < .005). Neither HME prevented contamination of the machine side of the circuit. These results support not reusing breathing circuits. Of 53 participants in group 2, 28 had positive ETT cultures with 7 showing transmission to anesthesia breathing circuit. Of 46 participants in group 1, 28 had positive ETT cultures with 9 showing transmission to anesthesia breathing circuit.

Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma tumors. I. Mitogen responses, mixed lymphocyte reactions (MLR) and mixed lymphocyte-tumor reactions (MLTR).

Impairment of mitogen responses to Con A and LPS and of MLR and MLTR was detected in the spleens of rats bearing syngeneic Moloney sarcoma tumors. Depressed responses of both T cell and Ig+ cell populations were observed. During the observation period of 6 to 10 days post-tumor inoculation when maximal T cell-mediated cytotoxicity was observed in spleen and draining lymph node cells, spleen cells showed marked impairment in response to stimuli mentioned above. By contrast, draining lymph node cell activity was either unaltered or somewhat elevated above the level of activity measured in normal control populations. Data presented in this and an accompanying paper strongly indicate that macrophages are activated as immunosuppressor cells in tumor-bearing rats.

Mosaic analysis of the dominant mutant, Gnarley1-R, reveals distinct lateral and transverse signaling pathways during maize leaf development.

Maize leaves are organized into two major domains along the proximal-distal axis: a broad flat blade at the distal end of the leaf, and a narrow, thickened sheath that encircles the stem. Between the blade and sheath are two wedge-shaped tissues called auricles, and the ligule, an epidermally derived fringe. Members of the Knotted1 (Kn1) family of mutations change the shape and position of both ligule and auricle, thus disturbing the overall pattern of the leaf. Here we present the results of a mosaic analysis of Gnarley1-R (Gn1-R), which like members of the Kn1 family, affects the ligule and auricle. Gn1-R is distinct, however, in altering the dimensions of cells that make up sheath tissue. To gain insight into the Gn1-R phenotype, we performed a mosaic analysis using X-ray induced chromosome breakage to generate wild-type (gn1+/-) sectors in otherwise Gn1-R leaves. These sectors allowed us to determine whether Gn1-R acts non-autonomously to influence adjacent cells. Most aspects of the Gn1-R phenotype, such as ligule position, inhibition of auricle development, and sheath thickness showed autonomy in the lateral dimension (leaf width). In contrast, all aspects of the Gn1-R phenotype were non-autonomous in the transverse dimension (leaf thickness), suggesting that signaling occurs between cell layers in the leaf. These results support a model for distinct signaling pathways along lateral versus transverse axes of a developing leaf.

Salivary epidermal growth factor in patients with and without acid peptic disease.

Epidermal growth factor inhibits gastric acid secretion and has a cytoprotective effect on the upper gastrointestinal tract. This study was undertaken to determine whether patients with endoscopically proven active peptic ulcer disease have a salivary deficiency of human epidermal growth factor (hEGF), compared to patients with a normal esophagogastroduodenoscopy (EGD). Saliva was collected from fasting subjects prior to EGD. The levels of EGF were measured by radioimmunoassay. Statistical evaluation was performed by analysis of variant followed by Student's t test. The concentrations of the peptide were lower in patients with active peptic ulcer disease (3.1 +/- 0.54 ng/ml, mean +/- SE, n = 25) compared with normal subjects (4.9 +/- 0.56 ng/ml, n = 58, p less than 0.03). No significant differences in salivary hEGF were noted between patients with a normal EGD and patients with gastritis (3.85 +/- .86 ng/ml, n = 13), esophagitis (4.5 +/- 1.3 ng/ml, n = 7), or Barrett's esophagus (5.3 +/- 1.5 ng/ml, n = 6). There were no differences in the salivary levels of hEGF between males and females, or between smokers and nonsmokers. There was no correlation of hEGF levels with age. The pathophysiologic significance of this finding is uncertain. Lower salivary hEGF may reduce one of the defensive mechanisms responsible for protecting the gastroduodenal mucosa from injury by physicochemical agents, thus contributing to ulcer development.

Cell-mediated cytotoxicity to moloney sarcoma in syngeneic and allogeneic rats.

Cell-mediated cytotoxicity in the primary immune response to Moloney sarcoma tumor (MST) in allogeneic and syngeneic rats was found to be predominantly T-cell-dependent. A minor non-T-cell cytotoxic activity may also have been detected. CMC was presumably directed against tumor and viral related antigens in the syngeneic host and primarily against alloantigens in the allogeneic host. CMC was more virgorous in the syngeneic host. This may be due to differences in quantities or immunogenicities of the various antigens involved. Two peaks of T-cells in effector populations were observed during a 20-day post-inoculation period. The first peak corresponded to peak T-cell-mediated cytotoxicity on day 8 and the second peak occurred on days 13 or 14 when CMC was minimal or undetectable.

[Modulation of graft versus host disease by in vitro incubation of donor cells with deoxycoformycin and deoxyadenosine]. / Modulation der Graft-versus-host-Erkrankung durch In-vitro-Inkubation von Spenderzellen mit Deoxycoformycin und Deoxyadenosin.

The combination of deoxycoformycin and deoxyadenosine was investigated for its capability to deplete T-cells from bone marrow and spleen cells and for its effect on GVHD in MHC-mismatched transplantation in rats. In vitro incubation with DCF/dADO for 18-20 hours resulted in significant but incomplete T-cell depletion without toxicity towards CFU-M. This corresponded with a lower incidence and a modification of GVHD following transplantation of such treated cells into MHC-incompatible recipient rats. However, GVHD could not be completely prevented by the in vitro treatment of donor cells.