Expression of inducible nitric oxide synthase in a mouse model of anaphylaxis.

BACKGROUND: The generation of large quantities of nitric oxide (NO) is implicated in the pathogenesis of anaphylactic shock. The source of NO, however, has not been established and conflicting results have been obtained when investigators have tried to inhibit its production in anaphylaxis. OBJECTIVE: The aim of this study was to analyze the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in a mouse model of anaphylaxis. METHODS: BALB/c mice were sensitized and challenged with ovalbumin to induce anaphylaxis. Tissues were removed from the heart and lungs, and blood was drawn at different time points during the first 48 hours after induction of anaphylaxis. The Griess assay was used to measure nitric oxide generation. Nitric oxide synthase expression was examined by reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: A significant increase in iNOS mRNA expression and nitric oxide production was evident as early as 10 to 30 minutes after allergen challenge in both heart and lungs. In contrast, expression of eNOS mRNA was not altered during the course of the experiment. CONCLUSION: Our results support involvement of iNOS in the immediate physiological response of anaphylaxis.

Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation.

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.

Reengineering a cardiovascular surgery service.

BACKGROUND: Reengineering, involving the radical redesign of business processes, has been used successfully in a variety of health care settings. In 1994 New York University (NYU) Medical Center (MC) launched its first reengineering team, whose purpose was to redesign the entire process of caring for patients-from referral to discharge-on the cardiovascular (CV) surgery service. REENIGINEERING TEAM: The multidisciplinary CV Surgery Reengineering Team was charged with two goals: improving customer (patient, family, and referring physician) satisfaction and improving profitability. The methodology to be used was based on a reengineering philosophy-discarding basic assumptions and designing the patient care process from the ground up. THE TRANSFER-IN INITIATIVE: A survey of NYU cardiologists, distributed in April 1994, suggested that the organization was considered a difficult place to transfer patients. The team's recommendations led to a new, streamlined transfer-in policy. The average waiting time from when a referring physician requested a patient transfer and the time when an NYUMC physician accepted the transfer decreased from an average of 9 hours under the old system to immediate acceptance. OTHER INITIATIVES: Three customer satisfaction task forces implemented multiple programs to make the service more user friendly. In addition, referrals increased and length of stay decreased, without an adverse impact on the mortality rate. CONCLUSION: For the first time at NYUMC, a multidisciplinary team was given the mandate to achieve major changes in an entire patient care process. Similar projects are now underway.

Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltage-gated K+ channels in rat spinal cord.

Prolonged opiate administration leads to the development of tolerance and dependence. These phenomena are accompanied by selective regulation of distant cellular proteins and mRNAs, including ionic channels. Acute opiate administration differentially affects voltage-dependent K+ currents. Whereas, opiate activation of K+ channels is well established opioid-induced inhibition of K+ conductance has also been studied. In this study, we focused on the effect of chronic morphine exposure on voltage-dependent Shaker-related Kv1.5 and Kv1.6 K+ channel gene expression and on Kv1.5 protein levels in the rat spinal cord. Several experimental approaches including in-situ hybridization, RNAse protection, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were employed. We found that motor neurons are highly enriched in Kv1.5 and Kv1.6 mRNA and in Kv1.5 channel protein. Moreover, we found significant increases in the amount of mRNA encoding for these two K+ channels and in Kv1.5 channel protein in the spinal cord of morphine-treated rats, compared with controls. For example, quantitative in-situ hybridization, revealed a 2.1 +/- 0.15- and 2.3 +/- 0.5-fold increase in Kv1.5 and Kv1.6 channel mRNA levels, respectively. Similar results were obtained by semiquantitative RT-PCR analyses. Kv1.5 protein level was increased by 1.9-fold in the spinal cord or morphine-treated rats. Our results suggest that Kv1.5 and Kv1.6 Shaker K+ channels play an important role in regulating motor activity that increases in mRNA and protein levels of the spinal cord K+ channels after chronic morphine exposure could be viewed as a cellular adaptation which compensates for a persistent opioid-induced inhibition of K+ channel activity. These alterations may account, in part, for the cellular events leading to opiate tolerance and dependence.

Modulation of the cellular phenotype by integrated adeno-associated virus.

The adeno-associated virus (AAV) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990, Microbiol. Rev. 54, 316-329). To investigate the expression of the AAV rep gene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neo hybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV and neo DNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418R cell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAV rep DNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplification in vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418R by AAV/neo DNA constructs with a deleted rep gene, or by a neo DNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAV rep gene interferes with cellular processes connected with DNA repair and gene amplification.

Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line.

Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.

Carcinogen-induced trans activation of gene expression.

We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

[Simple method of recording the kinetics of cell agglutination and aggregation]. / Prostoi metod registratsii kinetiki aggliutinatsii i agregatsii kletok.

The light scattering measure method for studying the kinetics of cell agglutination and aggregation under conditions of continuous and uniform stirring of suspension has been suggested.

[Kinetic method of registering cell agglutination in suspension]. / Kineticheskii metod registratsii aggliutinatsii keltok v suspenzii.

A method based on changes in cell light scattering during agglutination is proposed for registration of cell agglutination in suspension. Unlike other methods of investigation of cell agglutination, our measurements are made under constant stirring and minimal stress tension in specially constructed cells. Possibilities of the method are shown upon registration of agglutination of trypsinized rabbit erythrocytes caused by PHA, Ca ions or by a sample containing lectin isolated from the fresh water algae Chara. The sensitivity of our method is higher, than that with the microtitration according to Tacashi, and makes it possible to investigate the agglutination process kinetics.

Carcinogen-mediated induction of SV40 DNA synthesis in SV40 transformed Chinese hamster embryo cells.

Exposure of SV40-transformed Chinese hamster embryo cells (line CO50) to a series of physical and chemical carcinogens (including activation-dependent and activation-independent varieties) resulted in the induction of viral DNA synthesis. The carcinogen mediated amplification of SV40 DNA was demonstrated by a highly sensitive in situ hybridization procedure for the detection of cells synthesizing SV40 DNA. Treatment of CO50 cells with an inhibitor of polycyclic hydrocarbon metabolism (7,8-benzoflavone) prior to the application of benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene prevented the induction of SV40 DNA synthesis, indicating that the induction depends upon the metabolic activation of these compounds. Non-carcinogenic hydrocarbons were inactive under this assay. Two different protocols for determining the inducing potential of a compound are presented. The properties of this test and its possible use as a short-term assay for potential carcinogens is discussed. The possibility that the induction of SV40 DNA synthesis is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.