Renal expression of Na+-phosphate cotransporter mRNA and protein: Effect of the Gy mutation and low phosphate diet

The X-linked Gy mutation is closely linked, but not allelic, to Hyp and is characterized by rickets, hypophosphatemia, decreased renal tubular maximum for phosphate (Pi) reabsorption (Tm_P) and a specific reduction in renal brush-border membrane (BBM) Na⁺-Pi cotransport. Gy mice, like their normal littermates, respond to a low-Pi diet with an increase in BBM Na⁺-Pi cotransport, but fail to show an adaptive increase in Tm_p. Using an antibody raised against the NH₂ terminal peptide of the rat renal-specific Na⁺-Pi cotransporter (NaPi-2) and a NaPi-2 cDNA probe, we examined the effect of the Gy mutation and low-Pi diet (0.03% Pi) on NaPi-2 protein and mRNA abundance. The reduction in BBM Na⁺-Pi cotransport in Gy mice (51 ± 5% of normal, P < 0.05) was associated with a decrease in NaPi-2 protein (46 ± 12% of normal, P < 0.05) and mRNA abundance (76 ± 5%, P < 0.05). The low-Pi diet elicited a two- to three-fold increase in Na⁺-Pi cotransport in both normal and Gy mice that was accompanied by a large increase in NaPi-2 protein (10.2-fold in normal and 16.9-fold in Gy mice) and a modest increase in NaPi-2 mRNA (1.3-fold in both mouse strains, P < 0.05). The present data demonstrate that (1) the renal defect in BBM Pi transport in Gy mice can be ascribed to a deficit in NaPi-2 protein and mRNA abundance, (2) both normal and Gy mice respond to low Pi with an adaptive increase in NaPi-2 protein that exceeds the increase in Na⁺-Pi cotransport activity and NaPi-2 mRNA, (3) the adaptive increase in NaPi-2 protein and mRNA are not sufficient for the overall increase in Tm_P following Pi restriction. Received: 27 October 1995 / Received after revision: 4 December 1995 / Accepted: 6 December 1995     

Evaluation and standardization of polymerized ragweed extracts by chromatography,radioimmunoassay inhibition,and end point cutaneous titration

The standardization procedures for polymerized ragweed (PRW) must evaluate activity of PRW with assessments that differ from those used for standard unmodified extracts. This is because PRW allergens are different from conventional ragweed extracts in that they are much greater in average molecular weight and much lower in allergenicity for equivalent immunogenicity. We have evaluated seven samples of PRW for three parameters: allergenicity as determined by cutaneous end point titration, molecular weight distribution as determined by Sephadex G-200 chromatography, and availability of antigen E (AgE) determinants as measured by the ability of an extract to inhibit AgE binding to antibody by using a modification of the Farr technique. The skin test titers and molecular weight profiles provide information as to the safety of a PRW preparation and antigen-binding inhibitory activity gives information about allergenicity and immunogenicity. Appropriate limits may be set for each of these parameters to standardize PRW for clinical use.     

Loss of heterozygosity and mutational analyses of the ACTRII gene locus in human colorectal tumors

The activin type II receptorgene (ACTRII) is mutated in 58.1% of microsatellite-unstable (MSI-H) colorectal cancers and is a close relative of the TGFbeta-1 type II receptor, which is known to be involved in both MSI-H and non-MSI-H colorectal carcinogenesis. We therefore sought to determine whether ACTRII was involved in non-MSI-H colorectal cancers. We evaluated ACTRII inactivation by allelic deletion, loss of mRNA expression, or somatic mutation in 51 non-MSI-H colon cancers. Loss of heterozygosity (LOH) at the ACTRII locus (2q23.1) was found in 9 (17.6%) of 51 primary tumors. Loss of ACTRII mRNA expression was seen in one (14.3%) of the seven LOH-positive primary tumors from which total RNA was available. We also performed DNA sequencing analysis of tumors showing LOH. One LOH-positive primary tumor exhibited a novel germline missense sequence alteration (amino acid substitution, 117 Ile to Phe) that was not found in 23 additional normal individuals, implying that this alteration is not a frequent polymorphism. We conclude that ACTRII is probably involved in both non-MSI-H and MSI-H colorectal carcinogenesis, but more frequently in the latter subgroup.     

A Topographic study of differences in the P300 between introverts and extraverts

This paper presents results of a study to establish a link between neurocognitive psychophysiological and psychological type data through the investigation of differences in topographic auditory event-related potential (AERP) (P300) patterns in strongly introverted (n = 17) and strongly extraverted ( = 16) high school males as identified by the Myers Briggs Type Indicator. Group data files were created for the auditory event related potential task and converted to ASCII form. Amplitude values were evaluated at each scalp site. Kruskal Wallis one way analysis of variance was performed to evaluate group differences. In processing of infrequent, target stimuli, the amplitude of the P300 waveform for introverts was higher than for extraverts. When processing for non-target stimuli was subtracted from target stimuli, statistical differences were found over nine central, parietal, and occipital sites. The findings support and extend theories of biologically-based and bio-psycho-social typology.     

Polyribosomes of cells abortively or productively infected with adenovirus,papovavirus, or their hybrid

Summary The polyribosomes of African green monkey kidney (GMK) cells have been characterized during productive and abortive infections with adenovirus type 2, simian papovavirus SV40, and the PARA (defective SV40)-adenovirus type 2 hybrid. An early increase in uptake of H³-uridine was followed by a progressive decrease in both the amount of polyribosomes and in uptake of the label in all three systems that involved an adenovirus. Thus, the polyribosome distribution patterns obtained from GMK cells abortively infected with adenovirus type 2 did not differ significantly in optical density profile or incorporation of H³-uridine from the distributions obtained from GMK cells productively infected with either adenovirus 2 and SV40 or PARA-adenovirus type 2.A decrease in the amount of polyribosomes was not evident when GMK cells were infected with SV40. During the productive cycle of SV40 in GMK cells, the fifth (pentamer) polysome peak enlarged as the infection progressed. This increase was reflected both in increased optical density and incorporated H³-uridine. Material from this peak reacted with antibody to SV40 capsid antigen in the complement-fixation test. Arabinofurano-sylcytosine, a DNA inhibitor that blocks SV40 replication at a step prior to capsid formation, inhibited the formation of this peak. Treatment of the cellular extracts with EDTA or ribonuclease did not shift this peak to a lighter part of the gradient, along with the other polyribosomes. Virus particles, some of which proved to be infective for monkey cells, were present in electron micrographs of the pentamer peak.     

The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection

Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.     

IL-4 induces IL-6 and signs of allergic-type inflammation in the nasal airways of nonallergic individuals

In addition to its more widely recognized role in promoting IgE synthesis, we speculate that interleukin-4 (IL-4) may modulate both allergic- and nonallergic-type inflammatory processes in the airway mucosa. We examined in vivo the effect of IL-4 on granulocyte and cytokine homeostasis in the nasal airways of nonallergic volunteers. Ten (N = 10) healthy subjects received nasal IL-4 (10 microg) or saline (0.9%) challenges on separate occasions. Nasal lavage was obtained before and 24 h after nasal challenges. We report that IL-4 induced a significant increase in IL-6 and produced elevated levels of eosinophils and neutrophils compared to saline. These data demonstrate that IL-4 can modulate both allergic- and nonallergic-type inflammatory responses in the nasal airways of nonallergic individuals.     

Defibrination of blood plasma for use in serological tests for syphilis

Syphilitic plasma can be salvaged from discarded blood donations and converted to serum by defibrination. Sixty-nine units of plasma were treated with a stock solution of 100 U of thrombin per ml in 1 M calcium chloride and then with a 10% (wt/vol) solution of kaolin. Fibrinogen concentrations detected in initial plasma samples ranged from 94 to 4970 mg/liter (mean, 2532 mg/liter) for samples that were reactive by the rapid plasma reagin circle card test (RPR) and from 314 to 2742 mg/liter (mean 1528 mg/liter) for samples that were not reactive by the RPR. The treated samples showed no measurable fibrinogen remaining after the defibrination process. In the nontreponemal RPR for syphilis, 86% of the treated plasma samples retained the same endpoint titer as that of the initial plasma sample. When the Treponema pallidum passive-particle-agglutination test was used, 98% retained the same reactivity. In the Captia Syphilis-G enzyme immunoassay, 89% of the treated samples demonstrated no change in reactivity index, and in the fluorescent treponemal antibody absorption test, 96% showed no reduction in fluorescence. Human sera containing antibodies to syphilis are used at the Centers for Disease Control and Prevention for the preparation of reference controls or as samples for proficiency testing. Finding reactive sera is becoming more difficult due to the general decline of syphilis cases in the United States. The decreasing availability of these sera can be alleviated by salvaging plasma and converting it to serum.