Biomedical subjects

K Weis

Publications and source records attributed to K Weis.

16 recordsLinked to original sources

PubMed1998

Cloning and characterization of hSRP1 gamma, a tissue-specific nuclear transport factor.

Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/ NTF2. The NLS receptor is a heterodimeric proteins consisting of subunits of approximately 60 and 97 kDa, which have been termed importin alpha/beta, karyopherin alpha/beta, or PTAC 58/ 97. Members of the 60-kDa/importin alpha subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/ importin beta subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1 gamma, a human importin alpha homologue. The hSRP1 gamma protein is around 45% identical to the previously identified human importin alpha homologues hSRP1 alpha/Rch1 and NPI/ hSRP1. hSRP1 gamma can form a complex with importin beta and is able to mediate import of a BSA-NLS substrate in an in vitro nuclear import system. Interestingly, hSRP1 gamma shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1 gamma in tissue-specific transport events is discussed.

Amino Acid Sequence↗

PubMed1997

Exportin 1 (Crm1p) is an essential nuclear export factor.

Nuclear protein export is mediated by nuclear export signals (NESs), but the mechanisms governing this transport process are not well understood. Using a novel protein export assay in S. cerevisiae, we identify CRM1 as an essential mediator of nuclear protein export in yeast. Crm1p shows homology to importin beta-like transport factors and is able to specifically interact with both the NES motif and the Ran GTPase. A mutation in the shuttling protein Crm1p affects not only protein export, but also mRNA export, indicating that these pathways are tightly coupled in S. cerevisiae. The presented data are consistent with the conclusion that Crm1p is a carrier for the NES-mediated protein export pathway. We propose CRM1 be renamed exportin 1 (XPO1).

Biological Transport↗

PubMed1996

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.

Binding Sites↗

PubMed1996

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import.

Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy-dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1-like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS-containing proteins when added to an in vitro nuclear transport assay. While full-length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery.

Amino Acid Sequence↗

PubMed1996

RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus.

Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have different defects; Q69L (glutamine 69 changed to leucine) is defective in GTP hydrolysis while T24N (threonine 24 changed to asparagine) is defective in binding GTP. Both protein and snRNP import are sensitive either to the presence of the two mutant Ran proteins, which act as dominant negative inhibitors of nuclear import, or to incubation with nonhydrolyzable GTP analogues. This demonstrates that there is a requirement for a GTPase activity for the import of U snRNPs, as well as proteins, into the nucleus. The dominant negative effects of the two mutant Ran proteins indicate that the pathways of protein and snRNP import share at lease one common component.

Amino Acid Sequence↗

PubMed1995

Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences.

Import of proteins into the nucleus is a two-step process, involving nuclear localization sequence (NLS)-dependent docking of the substrate at the nuclear envelope followed by translocation through the nuclear pore. A recombinant human protein, hSRP1 alpha, bound in vitro specifically and directly to substrates containing either a simple or bipartite NLS motif. hSRP1 alpha promoted docking of import substrates to the nuclear envelope and together with recombinant human Ran reconstituted complete nuclear protein import. Thus, hSRP1 alpha has the properties of a cytosolic receptor for both simple and bipartite NLS motifs.

Amino Acid Sequence↗

PubMed1995

Molecular analysis of the coiled body.

There is increasing interest in studying how specific metabolic activities within the nucleus are organised into functional domains. The best known example is the nucleolus where rRNA genes are transcribed and rRNA processed and assembled into ribosomal units. Other subnuclear domains have been known for many years through morphological studies but are only recently being analysed at the molecular level. Here we focus on an evolutionarily conserved nuclear domain, called the coiled body, which contains splicing snRNPs. We review recent literature on the coiled body and discuss a possible model for its biological function.

Amino Acid Sequence↗

PubMed1994

The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif.

We have identified a new human Ca(2+)-binding protein that is specifically localized in the endoplasmic reticulum (ER). The protein is termed ERC-55, i.e. ER calcium-binding protein of 55 kDa. ERC-55 is a single copy gene and is encoded by an approximately 1900-base mRNA, which shows a ubiquitous expression pattern. The ERC-55 protein comprises an amino-terminal signal sequence followed by six copies of the EF-hand Ca2+ binding motif. Ca2+ binding was demonstrated directly for recombinant ERC-55 using the 45Ca2+ overlay technique. The carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL) is required for retention of ERC-55 in the ER. Deletion of HDEL results in slow secretion into the medium. In pulse-chase experiments, approximately 50% of the HDEL deletion mutant is secreted, whereas no detectable secretion is observed with the wild-type protein. This represents the first example of an endogenous human protein that is retained in the ER by an HDEL rather than Lys-Asp-Glu-Leu (KDEL) carboxyl-terminal tetrapeptide. Comparative sequence analysis indicates that ERC-55, together with the recently identified protein reticulocalbin (Ozawa and Muramatsu, 1993), constitute a new subfamily of the EF-hand superfamily of Ca(2+)-binding proteins that are specifically located in the ER.

Amino Acid Sequence↗

PubMed1994

Retinoic acid regulates aberrant nuclear localization of PML-RAR alpha in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.

Autoantibodies↗

PubMed1994

Immunodetection of poly(A) binding protein II in the cell nucleus.

During the polyadenylation of pre-mRNA in vitro, poly(A) binding protein II (PAB II) binds to the growing poly(A) tail, stimulating its extension. The subcellular localization of PAB II was investigated with an antibody affinity-purified from rabbit serum raised against the purified protein. Immunofluorescence microscopy detected PAB II exclusively in the cell nucleus, both in a widespread staining and in more intensely stained "speckles." PAB II was excluded from the nucleoli. By electron microscopy, PAB II was also found almost exclusively in the nucleus, predominantly in clusters of interchromatin granules, likely corresponding to the speckles observed by immunofluorescence microscopy, and in perichromatin fibrils, which represent nascent transcripts and probably the sites of pre-mRNA processing. In addition, electron microscopy also detected PAB II in nucleoli. The distribution corresponds largely to that of other factors involved in the processing of pre-mRNA and is thus in agreement with the proposed role of the protein in polyadenylation.

Blotting, Western↗

PubMed1993

[Longitudinal study of bronchial hyperreactivity using the carbachol test with school children].

STUDY DESIGN: Bronchial hyperresponsiveness of 476 schoolchildren (10.8 +/- 2.3 years) was studied three times at 12 months' intervals. The cumulative dose of 400 micrograms carbachol was applied in 50 + 50 + 100 + 200 micrograms steps. A fall of FEV1 of at least 15% was regarded as positive reaction. The test was save, as no severe obstruction was observed, only three children withdrew because of unpleasant cough. RESULTS: Reactivity was observed in 19.1, 10.0, and 5.2% of children at the occasion of the first, second and third test (sensitivity/specificity for prevalence of physician diagnosed asthma: 70/83, 35/91, and 24/96%, respectively). Reactivity was age dependent (7-11 years: 35%, 12-16 years: 18%), not influenced by sex, and the relative risk to be reactive was 1.9 in children 2-3 weeks after a respiratory tract infection. CONCLUSION: For epidemiological purposes carbachol provocation test--like other unspecific bronchial provocation tests--is inappropriate as a single test to classify individuals as asthmatics.

Adolescent↗

PubMed1991

Prepaid versus traditional Medicaid plans: lack of effect on pregnancy outcomes and prenatal care.

Enrollment of Medicaid recipients into capitated, case-managed systems has been advocated as a method of controlling cost. We studied prenatal care and birth outcomes for women and children enrolled in Aid to Families with Dependent Children (AFDC) in two capitated programs in Santa Barbara, California and Jackson County, Missouri (Prepaid), compared with similar but fee-for-service comparison medical communities in Ventura County, California and St. Louis, Missouri (FFS). At the sites of care, 2,336 inpatient and 823 prenatal care records were abstracted. Women at all sites received fewer than the recommended number of prenatal visits. At no site did more than 40 percent of women receive prenatal care in the first trimester of pregnancy. Mean birth weight and proportion of children of low birth weight (less than 2,500 grams) were similar between the demonstration and comparison counties. Complications of pregnancy and cesarean section rates were also similar between demonstration and comparison counties. This study did not demonstrate a decreased quality of care provided to enrollees in capitated, case-managed Medicaid programs compared with fee-for-service. Basic prenatal care was provided only to some members of this population, regardless of the type of physician payment.

Adult↗

PubMed1990

Prepaid versus traditional Medicaid plans: effects on preventive health care.

Prepaid, case managed systems have been proposed as a method of controlling costs in Medicaid populations. We investigated the utilization of preventive services in two prepaid Medicaid Competition Demonstration programs in Santa Barbara County, Calif., and Jackson County, Mo. (containing the city of Kansas City). Care in the demonstration sites was compared with care given in similar counties functioning under a traditional fee-for-service Medicaid system--Ventura County, Calif., and St Louis, Mo. We tested the hypothesis that preventive care would be less in the capitated demonstrations. 2735 Children's and 3389 adult's charts were abstracted for care received during the calendar year 1985, after the prepaid demonstration had been in place for more than 1 year. No significant differences were found between the demonstration and comparison counties in the proportion of children with complete DPT or OPV immunizations at 1 year of age, with 56% complete in both California counties and 69 and 65% complete in Jackson County and St Louis, respectively. Regression analysis demonstrated a slight, but statistically significant trend towards more immunizations in the demonstration counties. Pap smear use in women of 15-44 years of age was little different in the California counties, but significantly greater in the Jackson County demonstration in Missouri (64 vs 45%). Physician breast examinations were somewhat more likely to occur in the prepaid, case managed demonstration counties. Capitated, case managed systems for the AFDC Medicaid population appear to result in no diminution of preventive services. Substantial problems exist in this, as in other poor populations, in childhood immunizations.

Adolescent↗

PubMed1990

Diagnostic testing and return visits for acute problems in prepaid, case-managed Medicaid plans compared with fee-for-service.

Enrollment of Medicaid recipients into capitated, case-managed systems of health care has been advocated as a means to control costs. We studied the effect of such systems on care for urinary tract infection (UTI), pelvic inflammatory disease, and vaginitis among women enrolled in Aid to Families with Dependent Children in capitated demonstration programs in Santa Barbara County, California, and Jackson County, Missouri (prepaid), compared with similar but fee-for-service (FFS) counties in Ventura County, California, and St Louis, Mo. Structured abstracts were performed on 2382 outpatient charts with one of the three conditions in 1985. The proportion of cases with UTI in which a urine culture was obtained was similar in Santa Barbara (prepaid) and Ventura (FFS), 47% vs 46%, but greater in Jackson County (prepaid) than St Louis (FFS), 58% vs 32%. The proportion of cases with return visits for a UTI was: Santa Barbara (prepaid), 40%; Ventura (FFS), 33%; Jackson (prepaid), 72%; and St Louis (FFS), 53%. The proportion of patients with pelvic inflammatory disease with cervical cultures for gonorrhea as Santa Barbara (prepaid), 81%; Ventura (FFS), 52%; Jackson (prepaid), 86%; and St Louis (FFS), 61%. The proportion of women returning for follow-up after pelvic inflammatory disease was similar across all counties at 40% and 50%. The number of office diagnostic tests performed for vaginitis was greater in both demonstration counties. These data do not demonstrate any diminution in either diagnostic testing or follow-up visits for three common ambulatory problems in a Medicaid population enrolled in a capitated, case-managed system, with some trends for more care in the demonstration sites.

Adult↗

PubMedDate not supplied

Determination of serum oxalate using peroxyoxalate chemiluminescence of free oxalic acid.

We describe a new sensitive and specific method for determination of oxalate in human serum. By using the chemiluminescence decay of monoperoxyoxalic acid very low concentrations of oxalate (200 nmol/L) can be determined. The mean serum oxalate level in apparently healthy controls was 14.5 +/- 8.5 mumol/L. Supplementation of ascorbic acid leads to an increase in serum oxalate level. While serum oxalate concentrations of calcium oxalate stone formers (x = 16.4 +/- 9.8 mumol/L) are not significantly different from the control group, an extreme increase of serum oxalate is evident in haemodialysis patients. The serum oxalate concentration decreased during dialysis treatment from 141.4 +/- 32.1 mumol/L to 36.4 +/- 12.7 mumol/L.

Ascorbic Acid↗

PubMedDate not supplied

NCTR computer systems designed for toxicological experimentation. II. Experiment start-up system.

The Experiment Start-Up System (ESS) developed and implemented at the National Center for Toxicological Research constructs models from information contained in experimental protocols. These models may then be used to project resource requirements and related schedules. Each experimental model provides information from which management can determine the raw resources such as quantity of food, water, bedding, cages, animals (sex, strain, species), chemical, facilities, and manpower needed for conducting the experiment. Based upon this information, management can determine if the required resources are available and determine the most feasible "start" date for the new experiment. In addition, the information provided by the experimental model assures the Principal Investigator that the protocol requirements are thoroughly understood by participating technical support groups and that quality control elements of the ESS and other integrated systems will provide a means for accurately monitoring the experiment.

Computers↗