Disruption of PTPRO causes childhood-onset nephrotic syndrome

Idiopathic nephrotic syndrome (INS) is a genetically heterogeneous group of disorders characterized by proteinuria, hypoalbuminemia, and edema. Because it typically results in end-stage kidney disease, the steroid-resistant subtype (SRNS) of INS is especially important when it occurs in children. The present study included 29 affected and 22 normal individuals from 17 SRNS families; genome-wide analysis was performed with Affymetrix 250K SNP arrays followed by homozygosity mapping. A large homozygous stretch on chromosomal region 12p12 was identified in one consanguineous family with two affected siblings. Direct sequencing of protein tyrosine phosphatase receptor type O (PTPRO; also known as glomerular epithelial protein-1 [GLEPP1]) showed homozygous c.2627+1G>T donor splice-site mutation. This mutation causes skipping of the evolutionarily conserved exon 16 (p.Glu854_Trp876del) at the RNA level. Immunohistochemistry with GLEPP1 antibody showed a similar staining pattern in the podocytes of the diseased and control kidney tissues. We used a highly polymorphic intragenic DNA marker-D12S1303-to search for homozygosity in 120 Turkish and 13 non-Turkish individuals in the PodoNet registry. This analysis yielded 17 candidate families, and a distinct homozygous c.2745+1G>A donor splice-site mutation in PTPRO was further identified via DNA sequencing in a second Turkish family. This mutation causes skipping of exon 19, and this introduces a premature stop codon at the very beginning of exon 20 (p.Asn888Lysfs*3) and causes degradation of mRNA via nonsense-mediated decay. Immunohistochemical analysis showed complete absence of immunoreactive PTPRO. Ultrastructural alterations, such as diffuse foot process fusion and extensive microvillus transformation of podocytes, were observed via electron microscopy in both families. The present study introduces mutations in PTPRO as another cause of autosomal-recessive nephrotic syndrome.     

Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.     

Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons

Sphingosine 1‐phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high‐density lipoprotein (HDL)‐associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease‐severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease‐severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6–8 hrs, whereas the apoM decrease was seen later at 12–24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.     

Monitoring of protein product formation during fermentation with fast protein liquid chromatography

A method is described using fast protein liquid chromatography (FPLC) for the monitoring of protein formation during fermentation. The procedure consists of centrifugation to recover the cells, sonication of the cells, centrifugation to remove cell debris, and analysis of supernatant on a column of Mono Q (a strong anion exchanger). Analysis of peak areas provides quantitative determination of product concentration. Maintenance and life of the Mono Q column is discussed. We find that FPLC is a convenient method for measuring products in cell homogenates because it gives rapid, highly resolved separations.     

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.     

The <Emphasis Type="Italic"> GATE </Emphasis>retrotransposon in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>: mobility in heterochromatin and aspects of its expression in germline tissues

A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.