Pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus".

A prevalent and distinctive infectious interstitial pneumonia (IIP) of immunocompetent laboratory rats was suspected to be caused by a putative virus, termed rat respiratory virus, but this was never substantiated. To study this disease, 2 isolators were independently populated with rats from colonies with endemic disease, which was perpetuated by the regular addition of naive rats. After Pneumocystis was demonstrated by histopathology and polymerase chain reaction (PCR) in the lungs of rats from both isolators and an earlier bedding transmission study, the relationship between Pneumocystis and IIP was explored further by analyzing specimens from 3 contact transmission experiments, diagnostic submissions, and barrier room breeding colonies, including 1 with and 49 without IIP. Quantitative (q) PCR and immunofluorescence assay only detected Pneumocystis infection and serum antibodies in rats from experiments or colonies in which IIP was diagnosed by histopathology. In immunocompetent hosts, the Pneumocystis concentration in lungs corresponded to the severity and prevalence of IIP; seroconversion occurred when IIP developed and was followed by the concurrent clearance of Pneumocystis from lungs and resolution of disease. Experimentally infected immunodeficient RNU rats, by contrast, did not seroconvert to Pneumocystis or recover from infection. qPCR found Pneumocystis at significantly higher concentrations and much more often in lungs than in bronchial and nasal washes and failed to detect Pneumocystis in oral swabs. The sequences of a mitochondrial ribosomal large-subunit gene region for Pneumocystis from 11 distinct IIP sources were all identical to that of P. carinii. These data provide substantial evidence that P. carinii causes IIP in immunocompetent rats.

Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

STIM1: a novel phosphoprotein located at the cell surface.

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Murine Stim1 maps to distal chromosome 7 and is not imprinted.

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects.

Characterization of a KRAB family zinc finger gene, ZNF195, mapping to chromosome band 11p15.5.

We report the cDNA sequence of the zinc finger gene, ZNF195, which maps to chromosome 11p15.5. ZNF195 contains an N-terminal KRAB domain and 14 tandemly repeated Krüppel type zinc finger motifs at its C-terminus. Northern analysis shows expression of ZNF195 in adult heart, brain, placenta, skeletal muscle, and pancreas with a predominant transcript size of 4.3 kb. There is little expression in adult lung, liver, and kidney. In fetal lung, liver, kidney, and brain, the predominant transcript is 3.5 kb. Fetal brain also expresses a 4.3-kb transcript. RT-PCR analysis shows that two exons, 4a, which contains an inverted Alu sequence, and 4b, are differentially spliced and absent from the major transcript.

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.

Molecular cloning of a novel human gene (D11S4896E) at chromosomal region 11p15.5.

A novel gene (GOK) has been cloned from human chromosome region 11p15.5 that is believed to contain a gene or genes associated with a number of pediatric malignancies, including Wilms tumor. A 4-kb cDNA has been cloned and it encodes a predicted protein of approximately 84 kDa that could be translated in vitro. Computer analysis predicted that the protein had a signal peptide and may contain a transmembrane helix. Restriction mapping by pulsed-field electrophoresis indicates that GOK is located 1.7 kb telomeric of RRM1, and both genes are transcribed in the same direction. GOK displays high evolutionary conservation: cloning and partial sequencing of a mouse genomic clone revealed 90% identity with the human gene at both the nucleotide and the predicted amino acid levels.

Human gene for the large subunit of ribonucleotide reductase (RRM1): functional analysis of the promoter.

Ribonucleotide reductase comprises two nonidentical protein subunits R1 and R2, both of which are required for enzyme activity and show cell cycle-dependent regulation. The TATA-less promoter of the human RRM1 gene (encodes R1 protein) was examined with reference to regulatory domains upstream of the transcription start site. A region from nt -195 to +3 was found to give maximal expression of a reporter gene when transfected into the human cell line K562. Overall, this 198-bp region shows 58% identity with the equivalent region of the murine promoter; however, it contains two 22-bp domains that were 81 and 91% identical between species. Electrophoretic mobility shift assays were performed using a fragment of the domain closest to the transcription start site. These experiments revealed that several factors were able to bind this region in a sequence-specific manner. One of these factors was shown to be Sp1 by specific competition and supershift using antibody to Sp1. The data presented suggest that Sp1 is involved in the transcription of RRM1.

Human R1 subunit of ribonucleotide reductase (RRM1): 5' flanking region of the gene.

Ribonucleotide reductase is essential, in dividing cells, for the production of deoxyribonucleotides prior to DNA synthesis in S phase. Neither of its two subunits (R1 and R2) are detectable in quiescent cells. In cycling cells, RRM1 mRNA and R1 protein are present throughout the cell cycle. A fragment of the human cDNA was used to isolate a genomic clone that encompasses the 5' flanking region of human RRM1. Primer extension and PCR experiments were used to define six potential cap sites. The immediate upstream region does not have a TATA box and is not GC-rich. A 1.9-kb fragment (-1670 to +208) was able to direct transcription of a reporter gene in a transient expression system. Understanding the mechanisms regulating expression of this gene will provide insight into the processes involved in cell cycling and may be of particular importance in understanding the deregulated growth of transformed cells.

Therapeutic management of chronic suppurative otitis media with otic drops.

The use of potentially ototoxic topical drugs is controversial. Few experimental reports of audiometric data from human subjects exist. The purpose of this study is to determine if a significant difference between bone conduction hearing sensitivity before and after otic drop treatment for children with chronic suppurative otitis media and patent ventilation tubes exists. A statistical analysis of sensorineural threshold status after treatment of chronic suppurative otitis media with otic drops is presented.

TSEN: a novel MNS-related blood group antigen.

We report an antibody (anti-TSEN) that recognizes an antigen (TSEN) at the unique amino acid sequence that results from the junction of GPA58 to GPB27 if the GPB carries the S antigen. Red cells from several unrelated donors that possess this specific GP(A-B) hybrid molecule were agglutinated by anti-TSEN. Since a synthetic peptide with the amino acid sequence at this junction (Pro-Glu-Glu-Glu-Thr-Gly-Glu-Met-Gly-Gln-Leu-Val-His-Arg) specifically inhibited anti-TSEN, it must detect an antigen within this novel amino acid sequence. The TSEN antigen has been provisionally assigned the MNS blood group system number 002.033 on behalf of the ISBT Working Party on Terminology for Red Cell Surface Antigens.

Phase I studies of C. parvum given intravenously: effects of dexamethasone on its biologic and toxic properties.

Three biologic effects were clearly associated with C. parvum infusions: a) granulocytosis with lymphomonocytopenia, b) increase in responsiveness to phytohemmagglutinin and c) increase of specific antibodies to C. parvum antigens. Addition of dexamethasone moderately altered the kinetics of leukocyte changes without affecting other properties. Side effects after intravenous administration of C. parvum (2.5 mg/m2) consisted primarily of chills and fever which lasted for periods of 6-24 hours. Side effects occurred in 77% of the infusions. Addition of dexamethasone (4 mg IVP) reduced significantly the frequency (only 22% of patients) and intensity (fever 102 F and chills for only 30-60 minutes) of side effects. Clearly, dexamethasone of the dosage employed did not abrogate C. parvum effects while reducing significantly its toxic properties.