Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

The relationship of mutations in alpha- and beta-tubulin to microtubule assembly and anti-mitotic drug resistance

The mechanisms responsible for anti-cancer drug (including Taxol) treatment failure have not been identified. In cell culture model systems, many β-tubulin, but very few α-tubulin, mutations have been associated with resistance to Taxol. To test what, if any, mutations in α-tubulin can cause resistance, we transfected a randomly mutagenized α-tubulin cDNA into Chinese hamster ovary (CHO) cells and isolated drug resistant cell lines. A total of 12 mutations were identified in this way and all of them were confirmed to confer Taxol resistance. Furthermore, all cells expressing mutant α-tubulin had less microtubule polymer. Some cells also had abnormal nuclei and enlarged cell bodies. The data indicate that α-tubulin mutations confer Taxol resistance by disrupting microtubule assembly, a mechanism consistent with a large number of previously described β-tubulin mutations. ^ Because α- and β-tubulin are almost identical in their three dimensional structure, we hypothesized that mutations discovered in one subunit, when introduced into the other, would produce similar effects on microtubule assembly and drug resistance. 9 α- and 2 β-tubulin mutations were tested. The results were complex. Some mutations produced similar changes in microtubule assembly and drug resistance irrespective of the subunit in which they were introduced, but others produced opposite effects. Still one mutation produced resistance when present in one subunit, yet had no effect when present on the other; and one mutation that produced Taxol resistance when present in α-tubulin, resulted in assembly-defective tubulin when it was present in β-tubulin. The results suggest that in most cases, the same amino acid modification in α- and β-tubulin affects the microtubule structure and assembly in a similar way. ^ Finally, we tested whether three β-tubulin mutations found in patient tumors could confer resistance to Taxol by recreating the mutations in a β-tubulin cDNA and transfecting it into CHO cells. We found that all three mutations conferred Taxol resistance, but to different extents. Again, microtubule assembly in the transfectants was disrupted, suggesting that mutations in β-tubulin are a potential problem in cancer therapeutics. ^

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

ANALYSIS OF β8 INTEGRIN IN NEUROGENESIS AND NEUROVASCULAR HOMEOSTASIS

Neurogenesis in the adult mouse brain occurs within the subventricular zone (SVZ) of the lateral ventricle. In the SVZ, neural stem cells (NSC) reside in a specialized microenvironment, or vascular niche, consisting of blood vessels and their basement membranes. Most NSCs in the SVZ differentiate into progenitor cells, which further differentiate to generate neuroblasts, which then migrate from the SVZ to the olfactory bulbs (OB) along the rostral migratory stream (RMS). ECM-mediated adhesion and signaling within the vascular niche likely contribute to proper NSC self-renewal, survival, differentiation and neuroblast motility. The mechanisms that control these events are poorly understood. Previous studies from our group and others have shown that loss of the ECM receptor, αvβ8 integrin, in NSCs in the embryonic mouse brain leads to severe developmental vascular defects and premature death. Here, the functions of αvβ8 integrin in the adult brain have been examined using mice that have been genetically manipulated to lack a functional β8 integrin gene. This study reveals that loss of β8 integrin leads to widespread defects in homeostasis of the neurovascular unit, including increased intracerebral blood vessels with enhanced perivascular astrogliosis. Additionally, β8 integrin dependent defects in NSC proliferation, survival, and differentiation, as well as neuroblast migration in the RMS were observed both in vivo and in vitro. The defects correlated, in part, with diminished integrin-mediated activation of TGFβ, an ECM ligand of β8 integrin. Collectively, these data identify important adhesion and signaling functions for β8 integrin in the regulation of neural stem and progenitor cells in the SVZ as well as in neuroblast migration along the RMS in the adult brain.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

Ca++/calmodulin-dependent protein kinase II: Messenger RNA in developing rat brain

Ca$\sp{++}$/calmodulin-dependent protein kinase II (CaM-KII) is highly concentrated in mammalian brain, comprising as much as 2% of the total protein in some regions. In forebrain, CaM-KII has been shown to be enriched in postsynaptic structures where it has been implicated in maintaining cytoskeletal structure, and more recently in signal transduction mechanisms and processes underlying learning and memory. CaM-KII appears to exist as a holoenzyme composed of two related yet distinct subunits, alpha and beta. The ratio of the subunits in the holoenzyme varies with different brain regions and to some degree with subcellular fractions. The two subunits also display distinct developmental profiles. Levels of alpha subunit are not evident at birth but increase dramatically during postnatal development, while levels of beta subunit are readily detected at birth and only gradual increase postnatally. The distinct regional, subcellular and developmental distribution of the two subunits of CaM-KII have prompted us to examine factors involved in regulating the synthesis of the subunit proteins.^ This dissertation addresses the regional and developmental expression of the mRNAs for the individual subunits using in situ hybridization histochemistry and northern slot-blot analysis. By comparing the developmental profile of each mRNA with that of its respective protein, we have determined that initiation of gene transcription is likely the primary site for regulating CaM-KII protein levels. Furthermore, the distinct cytoarchitecture of the hippocampus has allowed us to demonstrate that the alpha, but not beta subunit mRNA is localized in dendrites of certain forebrain neurons. The localization of alpha subunit mRNA at postsynaptic structures, in concert with the accumulation of subunit protein, suggests that dendritic synthesis of CaM-KII alpha subunit may be important for maintaining postsynaptic structure and/or function. ^

Molecular basis of retinoid action: The role of retinoic acid receptors in retinoic acid-induced tissue transglutaminase expression

Retinoic acid has profound effects on the cellular growth and differentiation of a variety of cells. However, the molecular basis of retinoic acid action has, until recently, not been well understood. The identification of retinoic acid receptors which bear a high degree of homology to members of the steroid receptor super-family has dramatically altered our understanding of the biology of retinoids. The focus of this dissertation has been toward identification of retinoic acid binding proteins responsible for the effects of this molecule on gene expression.^ We have characterized in detail the retinoic acid-dependent induction of tissue transglutaminase gene expression in a myeloid cell line, human promyelocytic leukemia cells (HL-60 cells). Using cDNA probes specific for tissue transglutaminase, we have determined that the retinoic acid induced increase in enzyme level is due to an increase in the level of tissue transglutaminase mRNA. We have used this model as a probe to investigate the molecular basis of retinoid regulated gene expression.^ This thesis demonstrates that retinoic acid receptors are expressed in cells which induce tissue transglutaminase expression in response to retinoic acid. In Hl-60 cells retinoic acid-induced transglutaminase expression is associated with saturable nuclear retonic acid binding. Transcripts for both the alpha and beta forms of the retinoic acid receptors can be detected in these cells. Pretreatment of HL-60 cells with agents that potentiate retinoic acid-induced transglutaminase expression also modestly induced the alpha form of the retinoic acid receptor. Studies in macrophages and umbilical vein endothelial cells have also associated expression of the beta form of the retinoic acid with retinoic acid induced tissue transglutaminase expression.^ To investigate directly if retinoic acid receptors regulate retinoic acid-induced tissue transglutaminase expression we developed a series of stably transfected Balb-c 3T3 cells expressing different levels of the beta or gamma form of the retinoic acid receptor. These studies indicated that either the beta or gamma receptor can stimulate endogenous tissue transglutaminase expression in response to retinoic acid. These are among the first studies in the steroid field to describe regulation of an endogenous gene by a transfected receptor. ^

Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^