This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray (PAG) and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund’s adjuvant (CFA). Matrix-Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1 (HK-1), which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the PAG and cuneiform nucleus. SubP content in homogenates of the PAG and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.

Background Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process.Objectives To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH.Methods Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3-/- mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH.Results In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3-/- FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3-/- FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3-/- FLS compared to ASIC3+/+ FLS.Conclusion ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.

The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 -/- mice and examined the primary (response to tweezers) and secondary hyperalgesia (von-Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 -/- mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 -/- mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene-related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co-localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.

Familial hypomagnesemia with secondary hypocalcemia (HSH) (MIM 307600) was studied in three inbred Bedouin kindreds from Israel. The three kindreds, one extended and two nuclear families, contained 13 affected individuals, 11 males and two females. Assuming that the individuals affected with hypomagnesemia shared a chromosomal region inherited from a common ancestor, we used a DNA pooling strategy in a genome-wide search for loci which show homozygosity for shared alleles in affected individuals. DNA samples from affected individuals within a single kindred were pooled and used as the template for PCR amplification of short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected siblings and parents were used as controls. A shift towards homozygosity was observed in the affected DNA pool compared with the control pools with D9S301 (GATA7D12). Genotyping of individual DNA samples with D9S301 and several flanking markers confirmed linkage to chromosome 9 with maximum LOD scores of 3.4 (theta = 0.05), 3.7 (theta = 0) and 2.3 (theta = 0) for the three families. We have identified a 14 cM interval on chromosome 9 (9q12-9q22.2), flanked by proximal marker D9S1874 and distal marker D9S1807, within which all affected individuals from the three kindreds are homozygous for a shared haplotype. The disease segregates with a common affected haplotype in the three families, suggesting that hypomagnesemia is caused by a common ancestral mutation in these families. Although HSH has been previously reported to be X linked, these linkage data demonstrate that the disorder is an autosomal recessive disease in these kindreds. Mapping of a chromosomal breakpoint in a somatic cell line established from a patient with HSH and a balanced X;9 translocation placed the chromosomal breakpoint in a 500 kb region flanked by D9S1844 and D9S273. Identification of the gene responsible for hypomagnesemia will provide insight into the regulation of this essential cation.

In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S(HSD)). To evaluate the genetic integrity and salt sensitivity of thse new S(HSD), we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S(HSD), 16 "authentic" SS/Jr from the inbred colony of John Rapp (S(Rapp)), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R(HSD)), and (genotyping only) 6 known "contaminated" Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S(Rapp) and S* and 18 were polymorphic between S(Rapp) and R(Rapp), but none of the 22 markers revealed polymorphisms between S(Rapp) and the new S(HSD). The phenotyping studies showed that during an ultra-low salt diet, mean arterial pressure was higher (P < .05) in both authentic S(Rapp) (129 +/- 2 mm Hg; mean +/- SE) and new S(HSD) (120 +/- 2 mm Hg) than in R(HSD) (93 +/- 1 mm Hg). A high salt diet increased mean arterial pressure in every S(HSD) and S(Rapp). Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P < 0.05) greater in authentic S(Rapp) (+51 +/- 3 mm Hg) than in new S(HSD) (+39 +/- 3 mm Hg). In addition, salt-induced mortality was significantly greater in S(Rapp) (62.5%) than S(HSD) (8.3%) after 8 weeks (P < 0.01). S(HSD) were genotypically indistinguishable from S(Rapp), had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S(HSD) supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S(HSD) compared with S(Rapp). This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S(HSD) and S(Rapp) may now constitute distinct substrains of Dahl SS/Jr.

This chapter discusses the preparation of intramolecularly cross-linked hemoglobins. Hemoglobin (Hb), although normally a tetramer, readily undergoes dissociation to form αβ dimers. By cross-linking the molecule intramolecularly, this process can be blocked. This has become important both for basic structure-function studies of Hb and for the development of Hb derivatives useful as blood substitutes. Preparation of mixed metal and valency hybrids has made it possible to isolate stable species representing intermediate ligation states of Hb; these have yielded important new insights into the linkage between ligand binding and the structural and energetic changes that underlie cooperativity. Cross-linking is also important from the standpoint of developing Hb derivatives to serve as blood substitutes. The αβ dimers having a molecular weight of only 32,000 are readily filtered from the circulation by the kidneys. As a result, unmodified Hb has a very short plasma half-life, and the massive hemoglobinuria that results poses the risk of renal injury. It is essential to cross-link the molecule intramolecularly to prevent this from occurring.

Cross-contamination with previously amplified products poses a serious limitation in the use of PCR for clinical testing and in certain research applications as well. In the present study we report the use of novel primers containing a 3'-terminal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating in any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and post-amplification sterilization can be carried out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, is also destroyed.