Biomedical subjects

A Lin

Publications and source records attributed to A Lin.

At least 37 records · Page 2Linked to original sources

PubMed2001

Results of 3-year phase III clinical trials with daclizumab prophylaxis for prevention of acute rejection after renal transplantation.

BACKGROUND: Daclizumab (Zenapax, Roche Pharmaceuticals), a humanized monoclonal antibody directed against the alpha chain of the interleukin 2 receptor, has been shown to reduce the incidence of acute rejection at 6 months after renal transplantation in two phase III clinical trials. This report presents the combined 1- and 3-year outcomes of kidney transplant recipients who participated in these two phase III clinical trials. METHODS: Data from two multicenter, randomized, placebo-controlled trials were evaluated with regard to graft survival, patient survival, incidence of malignancies (including lymphoma), renal function (serum creatinine and glomerular filtration rate [GFR]), and current maintenance immunosuppressive regimen. In addition, the impact of acute rejection and acute rejection requiring treatment with antilymphocyte therapy upon 3-year graft survival was evaluated. Daclizumab was compared to placebo on a background of cyclosporine (CsA), azathioprine, and corticosteroids (triple therapy, TT) or CsA and corticosteroids (double therapy, DT). RESULTS: Treatment with daclizumab in the pooled analysis demonstrated a significant reduction in the incidence of biopsy-proven acute rejection episodes at 12 months posttransplant (43% vs. 28%, P<0.001). The 3-year graft survival was not significantly different between placebo and daclizumab-treated patients in the TT trial (83% vs. 84%) or in the DT trial (78% vs. 82%). Pooled patient survival was excellent in both placebo- (91%) and daclizumab- (93%) treated patients. The incidence of malignancies or posttransplant lymphoproliferative disorder (PTLD) in placebo- versus daclizumab-treated groups was comparable in both clinical trials. Renal function was similar between placebo- and daclizumab-treated groups in both the TT and DT trials. The occurrence of delayed graft function, acute rejection requiring antilymphocyte therapy at 6 months, and acute rejection at 12 months posttransplant were associated with decreased graft survival rates at 3 years posttransplant. CONCLUSIONS: The beneficial effect of daclizumab prophylaxis upon the incidence of acute rejection after renal transplant with TT or with DT was not associated with adverse clinical sequelae, including the development of PTLD, at 3 years posttransplant. There was no beneficial effect of daclizumab on graft survival at 3 years, but the trial was inadequately powered to detect this. Both studies showed excellent graft and patient survival at 3 years.

Acute Disease↗

PubMed2001

Daclizumab (humanized anti-IL2Ralpha mAb) prophylaxis for prevention of acute rejection in renal transplant recipients with delayed graft function.

BACKGROUND: The purpose of this retrospective study was to determine the benefits of daclizumab, (Zenapax, Roche Pharmaceuticals) a humanized anti-interleukin-2Ralpha (IL-2Ralpha) monoclonal antibody, for prevention of acute rejection in renal transplant recipients with delayed graft function (DGF). METHODS: Data from two multicenter randomized placebo-controlled trials were pooled. DGF was defined by urine output <30 cc/hour, decline in serum creatinine of <0.5 mg/dl, or the need for dialysis within the first 24 hours after transplantation. RESULTS: At one year posttransplantation, the incidence of biopsy-proven acute rejection in patients with DGF was reduced from 44% in the placebo group to 28% in the daclizumab group. (P=0.03) Prophylaxis with daclizumab also delayed the onset of the first biopsy-proven acute rejection episode in patients with DGF from 29+/-43 days in the placebo group to 73+/-70 days in the daclizumab group. (P=0.004) The graft survival rates in patients with DGF at 1 year posttransplantation were 78% in the placebo group and 82% in the daclizumab treated group. (P=ns) Three patients in the placebo-treated group with DGF experienced graft loss due to acute rejection, whereas no patients in the daclizumab-treated group with DGF had graft loss due to acute rejection. The 1-year patient survival rate in those with DGF in the placebo and daclizumab groups were 93% and 98%, respectively. (P=ns) CONCLUSIONS: Daclizumab effectively reduced the incidence and delayed the onset of biopsy-proven acute rejection in this high-risk subgroup of patients with DGF after renal transplantation. Graft and patient survival rates were similar between placebo- and daclizumab-treated patients with DGF.

Acute Disease↗

PubMed2001

p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion.

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.

Adenoviridae↗

PubMed2001

The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex.

The eukaryotic ribosomal 5S RNA-protein complex (5S rRNP) is formed by a co-translational event that requires 5S rRNA binding to the nascent peptide chain of eukaryotic ribosomal protein L5. Binding between 5S rRNA and the nascent chain is specific: neither the 5S rRNA nor the nascent chain of L5 protein can be substituted by other RNAs or other ribosomal proteins. The region responsible for binding 5S rRNA is located at positions 35-50 with amino acid sequence RLVIQDIKNKYNTPKYRM. Eukaryotic 5S rRNA binds a nascent chain having this sequence, but such binding is not substantive enough to form a 5S-associated RNP complex, suggesting that 5S rRNA binding to the nascent chain is amino acid sequence dependent and that formation of the 5S rRNP complex is L5 protein specific. Microinjection of 5S rRNP complex into the cytoplasm of Xenopus oocytes results in both an increase in the initial rate and also in the extent of net nuclear import of L5. This suggests that the 5S rRNP complex enhances nuclear transport of L5. We propose that 5S rRNA plays a chaperone-like role in folding of the nascent chain of L5 and directs L5 into a 5S rRNP complex for nuclear entry.

Active Transport, Cell Nucleus↗

PubMed2001

Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes.

The transcription factor NF-kappaB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-kappaB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-kappaB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-kappaB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-kappaB was inhibited by expression of a "supersuppressor" IkappaBalpha mutant that is resistant to stimulation-induced degradation and a dominant negative IkappaB kinase (IKKbeta) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IkappaBalpha degradation in an IKK-dependent manner, suggesting that NF-kappaB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IkappaBalpha mutant or the dominant negative IKKbeta mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-kappaB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-kappaB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Angiotensin II↗

PubMed2001

Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers.

We have shown recently (B. A. Yoshida et al., Cancer Res., 59: 5483-5487) that mitogen-activated protein kinase kinase 4 (MKK4) can suppress AT6.1 rat prostate cancer metastases in vivo. Evaluation of the expression of components of the MKK4 signaling cascade showed a loss or down-regulation of expression of MKK4 or c-Jun, a downstream mediator of MKK4, in six of eight human prostate cancer cell lines. Given these findings, we next assessed whether MKK4 dysregulation occurs during the development of clinical prostate cancer. Immunohistochemical studies showed high levels of MKK4 expression in the epithelial but not the stromal compartment of normal prostatic tissues. In neoplastic tissues, a statistically significant, direct, inverse relationship between Gleason pattern and MKK4 was established. These results demonstrate that MKK4 protein is consistently down-regulated during prostate cancer progression and support a role for dysregulation of its signaling cascade in clinical disease. To test the possibility that down-regulation of MKK4 protein is the result of allelic loss, metastatic prostate cancer lesions were examined for loss of heterozygosity (LOH) within the MKK4 locus (D17S969). These studies showed a 31% (5 of 16) LOH of MKK4 that is not associated with coding region mutations, which suggests that the nucleotide sequence of the gene in the remaining allele is infrequently mutated.

Enzyme Activation↗

PubMed2001

Evidence for a common binding cavity for three general anesthetics within the GABAA receptor.

The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

Anesthetics↗

PubMed2001

FasL is important in costimulation blockade-resistant skin graft rejection.

BACKGROUND: Simultaneous blockade of the CD40 and CD28 costimulatory pathways is effective in prolonging allograft survival in murine and primate models. Recent data suggest that intact apoptotic pathways are crucial for the induction of hyporesponsiveness by costimulation blockade. We have studied the impact of fas/fasL signaling, an important T cell apoptotic pathway, on the effects of costimulation blockade. Methods. Wild type, lpr (fas deficient), and gld (fasL deficient), mice were used as donors and recipients in the murine skin graft model. Allograft survival was compared in untreated and costimulation blockade (500 microg anti-CD40L and 500 microg CTLA4-Ig, days 0, 2, 4, 6) treated recipients. In some recipients, CD4+ T cells were depleted using rat anti-murine CD4 (100 microg day -3, -2, -1, and weekly). RESULTS: gld mice treated with costimulation blockade enjoy a significantly greater increase in skin allograft survival than do wild-type mice. This effect is not replicated using lpr donors or recipients. Experiments in which CD4+ cells were depleted demonstrate that fasL is not necessary for CD8-mediated allograft rejection, and that depletion of CD4+ cells eliminates some of the survival advantage induced by costimulation blockade. CONCLUSIONS: FasL is not required for the establishment of costimulation blockade induced hyporesponsiveness, but rather appears to be required for normal costimulation blockade resistant rejection. Fas expression is not critical for costimulation blockade resistant rejection, suggesting that fasL may be interacting with other receptors. Further, it appears that CD4+ cells are important in the maintenance of allograft protection induced by costimulation blockade in this model.

Animals↗

PubMed2001

Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2.

Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.

Animals↗

PubMed2001

Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics.

gamma-Aminobutyric acid type A (GABA(A)) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA(A) receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA(A) receptor beta(2) subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the beta(2) subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the beta(2)(M286W) mutation resulted in a decrease in the 'cut-off' volume for propofol analog molecules to enhance GABA responses at GABA(A) alpha(1)beta(2)gamma(2s) receptors. This suggests that mutation of M286 in the GABA(A) beta(2) subunit alters the dimensions of a 'binding pocket' for propofol and related alkylphenol general anesthetics.

Amino Acid Sequence↗

PubMed2001

Passive immunization against nicotine prevents nicotine alleviation of nicotine abstinence syndrome.

Passive immunization against nicotine interferes with its locomotor and pressor effects. The current study determined whether immunization could prevent another nicotine action: the reversal of nicotine abstinence syndrome. IgG containing 4.4-5.6% nicotine-specific antibody was isolated from rabbits immunized with 3'-amino-methyl-nicotine conjugated to a carrier protein. Twenty rats were rendered dependent by 7 days of subcutaneous infusion of 3.15 mg/kg/day nicotine (expressed as the base). Upon termination of nicotine infusion, each rat was injected intraperitoneally with 150 mg of IgG from normal serum (n=13) or from nicotine antiserum (n=7). Twenty-two and one-half hours later, all rats were observed over 15 min for baseline nicotine abstinence signs. Two and one-half hours after baseline observations, seven of the 13 rats pretreated with control IgG and all seven rats pretreated with nicotine-specific IgG were then challenged by 0.12 mg/kg (sc) nicotine. The remaining six rats pretreated with control IgG were challenged with saline alone. All rats were then observed again for abstinence signs. Nicotine injection caused significantly less reduction of abstinence signs in the immunized rats. The nicotine effect in immunized rats was comparable to the saline effect in nonimmunized rats. Immunization also significantly reduced free serum nicotine concentration and nicotine distribution to the brain. These results raise the possibility that immunization might prevent nicotine consumption from relieving the discomforts of smoking cessation.

Analysis of Variance↗

PubMed2001

Blocking caspase-3-mediated proteolysis of IKKbeta suppresses TNF-alpha-induced apoptosis.

The transcription factor NF-kappaB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-kappaB activation. It is unknown how the protection conveyed by NF-kappaB is overridden during apoptosis. We report here that IkappaB kinase (IKK) beta was specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-alpha-induced apoptosis. Proteolysis of IKKbeta eliminated its enzymatic activity, interfered with IKK activation, and promoted TNF-alpha killing. Point mutations that abrogate IKKbeta proteolysis generated a caspase-resistant IKKbeta mutant, which suppressed TNF-alpha-induced apoptosis. Thus, our study demonstrates that TNF-alpha-induced apoptosis requires caspase-mediated proteolysis of IKKbeta.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Further improvements in laparoscopic donor nephrectomy: decreased pain and accelerated recovery.

Fear of postoperative pain is a disincentive to living donor kidney transplantation. Laparoscopic donor nephrectomy (LDN) was developed in part to dispel this disincentive. The dramatic increase in the number of laparoscopic donor nephrectomies performed at our institution has been in part due to the reduction in postoperative pain as compared to traditional, open donor nephrectomy. We sought to further diminish the pain associated with this surgical technique. The purpose of this study was to compare the efficacy of three different postoperative pain management regimens after LDN. All living kidney donors performed laparoscopically (n=43) between September 1998 and April 2000 were included for analysis. Primary endpoints included postoperative narcotic requirements and length of stay. Narcotic usage was converted to morphine equivalents (ME) for comparison purposes. Patients received one of three pain control regimens (group 1: oral and intravenous narcotics; group II: oral and intravenous narcotics and the On-Q pump delivering a continuous infusion of subfascial bupivicaine 0.5%; and group III: oral and intravenous narcotics and subfascial bupivicaine 0.5% injection). Postoperative intravenous and oral narcotic use as measured in morphine equivalents was significantly less in group III versus groups I and II (group III: 28.7 ME versus group I: 40.2 ME, group II: 44.8 ME; P<0.05). Postoperative length of stay was also shorter for group III (1.8 days) versus group I (2.5 days) and group II (2.9 days). LDN has been shown to be a viable alternative to traditional open donor nephrectomy for living kidney donation. We observed that the use of combined oral and intravenous narcotics alone is associated with greater postoperative narcotic use and increased length of stay compared to either a combined oral and intravenous narcotics plus continuous or single injection subfascial administration of bupivicaine. The progressive modification of our analgesic regimen has resulted in decreased postoperative oral and intravenous narcotic use and a reduction in the length of stay. We recommend subfascial infiltration with bupivicaine to the three laparoscopic sites and the pfannenstiel incision at the conclusion of the procedure to reduce postoperative pain. We believe this improvement in postoperative pain management will continue to make LDN even more appealing to the potential living kidney donor.

Adult↗

PubMed2001

Regulation of organelle membrane fusion by Pkc1p.

Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers. The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p. While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated. We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C. Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation.

Adenosine Triphosphatases↗

PubMed2001

Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C.

Administration of interferon (IFN) 3 times weekly in patients with chronic hepatitis C (CHC) is associated with low sustained responses, which may be, in part, related to this regimen's inability to maintain IFN concentrations sufficient to suppress viral replication. An enhanced IFN molecule produced by the covalent attachment of a branched 40-kd polyethylene glycol moiety to IFN alpha-2a (PEG[40kd] IFN alpha-2a) exhibits sustained absorption, a restricted volume of distribution, and reduced clearance compared with unmodified IFN alpha-2a. One hundred fifty-nine patients with CHC participated in a randomized, ascending-dose (45 or 90, 180, 270 microg) study comparing PEG(40kd) IFN alpha-2a administered once weekly with 3 MIU IFN alpha-2a administered 3 times weekly for 48 weeks to determine the most appropriate PEG(40kd) IFN alpha-2a dose for subsequent clinical trials. Efficacy was assessed by measuring hepatitis C virus (HCV) RNA following a 24-week treatment-free period. Sustained virological responses for PEG(40kd) IFN alpha-2a once weekly were 10% (45 microg; not significant), 30% (90 microg; P = .009), 36% (180 microg; P = .0006), and 29% (270 microg; P = .004), compared with 3% for the 3-times-weekly 3-MIU IFN alpha-2a regimen. The types and frequencies of adverse events and laboratory abnormalities were similar among all groups. In conclusion, once-weekly PEG(40kd) IFN alpha-2a was associated with a higher number of sustained virological responses compared with IFN alpha-2a 3 times weekly in patients with CHC, but had a similar safety profile. The 180-microg PEG(40kd) IFN alpha-2a dose appeared to be the optimal dose based on sustained virological response and its associated side-effect profile.

Adult↗

PubMed2001

Use of autologous radial artery for revascularization of hepatic artery thrombosis after orthotopic liver transplantation: case report and review of indications and options for urgent hepatic artery reconstruction.

Hepatic artery thrombosis (HAT) is the most common vascular complication after orthotopic liver transplantation (OLT) and has traditionally been managed with re-OLT. However, several reports have shown that urgent revascularization is frequently an effective means of graft salvage. This most often involves hepatic artery (HA) thrombectomy and thrombolysis, with reestablishment of arterial inflow through a donor iliac artery conduit based on the supraceliac or infrarenal aorta. We report a 46-year-old man who developed HAT 13 days after OLT after angiographic splenic artery embolization to reduce splenic artery steal. A suitable donor iliac artery was not available for arterial reconstruction and could not be obtained from neighboring transplant centers. The patient underwent urgent HA thrombectomy, intrahepatic arterial thrombolysis, and revascularization using an autologous radial artery (RA) conduit based on the supraceliac aorta. The patient is alive more than 1 year after revascularization, with normal liver function and documented flow in the arterial conduit by Doppler ultrasound and arteriography. He has not developed late biliary complications or adverse sequelae of RA harvest. Autologous RA can be safely and successfully used as an aortic-based arterial conduit in urgent revascularization of HAT after OLT. RA should be considered for use in HA revascularization if an adequate donor iliac artery is not available and other potential conduits are not usable or desirable. The availability of autologous RA expands the armamentarium of vascular conduits that can be used in HA revascularization and may help minimize re-OLT for otherwise potentially salvageable liver allografts.

Arterial Occlusive Diseases↗

PubMed2001

Gene structure for adenosine kinase in Chinese hamster and human: high-frequency mutants of CHO cells involve deletions of several introns and exons.

The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells. The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority <100 nt. The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb). A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH. The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH. In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large. In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster [2], Caenorhabditis elegans [4], Arabidopsis thaliana [10]). Some of the intron-exon junctions in these species are in the same positions as in mammals. The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta 3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome. These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s). We have also determined the nature of the genetic alterations in two of the class A AK(-) mutants of CHO cells, which are obtained at a very high spontaneous frequency (10(-3)-10(-4)) in this cell line. Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts. The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted. The endpoints of these deletions lie in the large introns within the AK gene.

Adenosine Kinase↗

PubMed2001

The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.

Adaptor Proteins, Vesicular Transport↗