Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family

Silver-stainable proteins (SSPs) are aspartic acid-rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxyncleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA-derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine-rich repeats (LRR) near the NH2-terminal region and acidic clusters at the COOH-terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen-mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag-NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.     

Identification of human and mouse GP-1, a putative member of a novel G-protein family

OBJECTIVES: To determine a) if serum morphine concentration changes during the first 3 hrs of extracorporeal membrane oxygenation (ECMO); and b) if absorption of morphine onto the membrane oxygenator is responsible for these changes. Also, morphine clearance during the first 5 days of ECMO was studied. DESIGN: Prospective, open-label study with consecutive patient enrollment. SETTING: Neonatal intensive care unit at a university-affiliated, children's hospital. SUBJECTS: Eleven neonates with severe persistent pulmonary hypertension of the newborn receiving continuous intravenous infusions of morphine sulfate and requiring ECMO. INTERVENTIONS: Blood samples were obtained from the subjects and ECMO circuits at predetermined time intervals. MEASUREMENTS AND MAIN RESULTS: Serum morphine concentration was determined using high-performance liquid chromatography. Morphine concentrations were no different from baseline at 5 mins, 1 hr, or 3 hrs after beginning ECMO. There was no significant difference in morphine concentration from samples taken immediately proximal and distal to the membrane oxygenator at 5 mins, 1 hr, and 3 hrs after the start of ECMO. Morphine clearance was calculated on days 1, 3, and 5 of ECMO. The mean value for morphine clearance was 11.7 +/- 9.3 (SD) ml/min/kg (range 2.6 to 34.5). CONCLUSIONS: The initiation of ECMO does not lead to a significant decrease in serum morphine concentration and there is no uptake of morphine onto the membrane oxygenator of the ECMO circuit. Morphine clearance for infants receiving ECMO is variable.     

Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses

PD-1, an Ig superfamily member, contains an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic tail. It is expressed in a minor fraction of CD4-CD8- normal thymocytes and induced in peripheral lymphocytes following activation. To assess the possible roles of PD-1 in the immune responses, PD-1-deficient (PD-1-/-) mice were generated by a gene-targeting strategy. PD-1-4- mice developed and grew normally. Although the thymus was apparently normal, PD-1-/- mice showed moderate but consistent splenomegaly, which reflected the increased cellularity of both lymphoid and myeloid cells. The proliferative response of B cells by anti-IgM antibodies, but not of T cells by an anti-CD3 (145-2C11) mAb in vitro, was augmented in PD-1-/- mice as compared with control littermates. PD-1-/- mice showed increased serum levels of IgG2b, IgA and most strikingly IgG3, while those of IgM and IgG1 were comparable with control mice. Furthermore, PD-1-/- mice exhibited significantly augmented IgG3 anti-DNP antibody response to a type 2 T-independent antigen, DNP-Ficoll, with comparable IgM and IgG1 antibody responses with littermate controls. In the peritoneal cavity, the B-1 cell population in PD-1-/- mice exhibited significantly reduced expression of CD5, a negative regulator of B-1 cell activation, despite a marginal increase in the number of B-1 cells. Thus, PD-1 was suggested to be involved in the negative regulation for particular aspects of B cell proliferation and differentiation including class switching.     

ATP1AL1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump

The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

Identification of 21 novel human protein kinases, including 3 members of a family related to the cell cycle regulator nimA of Aspergillus nidulans

The nimA gene encodes a protein-serine/threonine kinase that is required along with the p34cdc2 kinase for mitosis in Aspergillus nidulans. We have searched for human protein kinases that are related to the NIMA protein kinase using the polymerase chain reaction. Different pairs of degenerate oligonucleotides specific for conserved amino acid motifs in the catalytic domain of NIMA were used as primers in the polymerase chain reaction to amplify partial complementary DNAs (cDNAs) of protein kinases expressed in the promyelocytic leukemia cell line HL-60. Forty-one distinct cDNAs representing a broad spectrum of serine/threonine- and tyrosine-specific protein kinases were identified, and the sequences for 21 of these protein kinases were found to be unique. Three of these cDNAs represent a family of protein kinases whose members are related to NIMA and the murine nimA-related protein kinase Nek1. We discuss the success of this polymerase chain reaction approach with respect to the use of multiple primer pairs, the influence of primer degeneracy, and the tolerance of cDNA amplification to mismatches between primers and template mRNA.     

Cabin 1, a negative regulator for calcineurin signaling in T lymphocytes

Rectus femoris surgery was performed on 70 patients with cerebral palsy and stiff-knee gait. Fifty-three patients underwent distal rectus transfer, and 17 patients had distal rectus release with complete muscle mobilization. Gait analysis was performed preoperatively and postoperatively at a minimum of 1 year. Swing-phase peak knee flexion (PKF) was improved in the transfer group, allowing improved foot clearance and more efficient gait (p = 0.04). PKF in swing deteriorated slightly in the release group (p = 0.04). The presence of abnormal swing-phase electromyogram (EMG) activity in the rectus alone or abnormal combined rectus and vastus lateralis activity did not influence the PKF results in either surgery (p < 0.05). The Ely test had no predictive value in identifying patients with abnormal EMG activity (p > 0.05). Preoperative knee range of motion was not a significant variable in determining relative success of rectus surgery. No deleterious effects were observed in stance phase in either group (p > 0.05).     

Identification and characterization of a novel member of the EXT gene family, EXTL2

Recently, two homologous genes, EXT1 and EXT2, with a putative tumor suppressor function have been described. Mutations in both genes are responsible for multiple exostosis syndrome (EXT), an autosomal dominant condition characterized by the presence of multiple osteochondromas, bony excrescences that sometimes undergo malignant transformation to chondrosarcoma. This family of EXT genes has been extended by the identification of an EXT-like (EXTL) gene showing a high degree of homology with the EXT genes. We report here a second EXT-like gene (EXTL2) which is homologous to the EXT and EXTL genes. EXTL2 consists of 5 exons encoding an ubiquitously expressed protein of 330 amino acids. In addition, a putative pseudogene, EXTL2P was also identified. The EXTL2 gene was assigned to chromosome 1p11-p12, whereas EXTL2P was mapped on chromosome 2q24-q31.     

StgR, a new Streptomyces alboniger member of the LysR family of transcriptional regulators

Many of the current tuberculosis control programmes in the Russian Federation are based on costly strategies which are underfunded and use long, individualized treatment regimens. This article compares, using a cost-effectiveness analysis, the new WHO strategy implemented in the Ivanovo Oblast (case-finding among symptomatic patients (SCF) and shorter regimens) and the old strategy (active screening of the asymptomatic population (ACF) and longer regimens). The cost per case cured was calculated at different levels of cure rate (45-95%) using three scenarios to describe the new WHO strategy (use of WHO-recommended regimens and three options at increasing rates of admission) and a fourth scenario to describe the old strategy (all patients admitted for the whole treatment and longer regimens). The cost per case detected was determined by calculating the following: yield of the new and old strategy (number of examinations necessary to diagnose one case); cost of the diagnostic process; multiplying yield per cost according to the three scenarios describing the new WHO strategy and a fourth scenario describing the old strategy. In the Ivanovo Oblast the cost per case cured, at 85% cure rate level, ranged from US$ 1197 (new strategy, scenario 1 without food) to US$ 6293 (old strategy, scenario 4) the cost per case detected ranged from US$ 1581 (new strategy, scenario 1) to US$ 4000 (old strategy, scenario 4). Significant savings can result from shifting towards the new WHO strategy. Decision-makers and health administrators should be responsible for re-investing the financial and human resources mobilized by the adoption of cost-effective strategies within the TB control programme.     

Identification of a novel member of the pentraxin family in Xenopus laevis

Pentraxins are a family of acute phase reactants. Two family members, C-reactive protein (CRP) and serum amyloid P component (SAP), are known in a range of mammalian species. CRP and SAP are both about 200 residues long, and arose from a gene duplication event, apparently before the divergence of the mammalian orders. To elucidate the origins of mammalian pentraxins, we have searched for pentraxin-coding genes in the amphibian Xenopus laevis. We have identified a gene determining a protein (XL-PXN1) which is about twice the size expected: the XL-PXN1 gene appears to be a fusion between regions encoding an amino-terminal peptide of unknown function and a carboxy-terminal pentraxin. The pentraxin domain is more divergent from CRP and SAP than they are from each other: it provides an outgroup for analysis of the evolution of mammalian pentraxins and confirms that putative CRP and SAP proteins partly characterized in non-vertebrate species cannot be true homologues of the mammalian proteins.     

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.