A specific and sensitive radioimmunoassay for rat C-peptide

A sensitive and specific radioimmunoassay for rat serum C-peptide (RCP) has been developed and validated using a guinea pig anti-rat C-peptide antibody to synthetic rat C-peptide. Negligible crossreactivity (<0.01%) to human proinsulin was observed, whereas human insulin, human pancreatic polypeptide (hPP), porcine insulin, porcine C-peptide, bovine insulin, rat insulin, porcine-PP, and glucagon, respectively, did not produce measurable displacement of RCP tracer. Human C-peptide even in a supraphysiological concentration range crossreacted poorly (<0.1%). The sensitivity limit of the assay calculated at ±3 standard deviations was 24.2pM (0.07 ng/mL). RCP standard concentrations ranged from 25–1600pM. The intraassay-and between assay-coefficient of variations (CV) were 3.5–6.1% and 4.1–9.5%, respectively. The mean percentage recovery of RCP added to rat serum samples was 100.8±2%. Serum volume dilution from 25 to 100 μL did not significantly alter the expected RCP level. Migration of rat serum C-peptide and that of synthetic RCP were identical in a Sephadex G-50 chromatographic analysis. The mean fasting and postprandial plasma RCP levels in normal rats were 102±15pM and 485±75pM, respectively. RCP levels following intravenous glucose tolerance test in diabetic and nondiabetic rats were consistent with expected patterns. In conclusion, we have developed and validated a rat C-peptide assay that is sensitive, simple, and specific for RCP in serum. The assay provides a reliable tool for studies of diabetes using rodent animal models.     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Cloning and expression of a Streptococcus sanguis surface antigen that interacts with a human salivary agglutinin.

Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.     

Immunity to vaginal reinfection in female guinea pigs infected sexually with Chlamydia of guinea pig inclusion conjunctivitis.

Guinea pig boars were inoculated intraurethrally with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). At the heights of their urethral infections, they were caged with sows in estrus. Whereas some of the sows had not been previously exposed to GPIC agent, others had received an intravaginal inoculation 5 to 8 weeks earlier. Those sows for which infected boars provided the first exposure were challenged by intravaginal inoculation 5 to 8 weeks later. Vaginal and conjunctival scrapings were taken regularly and stained for chlamydial inclusions. Titers of serum anti-GPIC antibodies and of vaginal secretory IgA anti-GPIC antibodies were determined by immunofluorescence. Our results show for the first time that a sexually acquired vaginal GPIC infection induces immunity to manual reinfection of the vagina. Because of the high incidence of secondary conjunctival infections among the vaginally infected sows, we could not provide a sound statistical basis for our tentative conclusion that manual infection of the vagina induces immunity to sexual reinfection. The results of our antibody titrations confirm previous work showing that vaginal GPIC infection induces formation of both serum antibody and vaginal secretory immunoglobulin A antibody.     

Developmental expression of FOG-1/CPEB protein and its control in the Caenorhabditis elegans hermaphrodite germ line.

The specification of a germ cell as sperm or oocyte and determination of cell number remain unsolved questions in developmental biology. This paper examines Caenorhabditis elegans FOG-1, a CPEB-related RNA-binding protein that controls the sperm fate. We find that abundant FOG-1 protein is observed transiently in germ cells just prior to their expression of an early sperm-differentiation marker. As the germline tissue elongates, abundant FOG-1 appears more and more distally as sperm become specified, but disappears when the germ line switches to oogenesis. This dynamic pattern is controlled by both globally acting and germline-specific sex-determining regulators. Importantly, the extent of FOG-1 expression corresponds roughly to sperm number in wild-type and mutants, altering sperm number. By contrast, three other key regulators of the sperm/oocyte decision do not similarly correspond to sperm number. We suggest that FOG-1 is precisely modulated in both time and space to specify sperm fate and control sperm number.     

A locus for Bowen-Conradi syndrome maps to chromosome region 12p13.3

Bowen-Conradi syndrome (BCS) is a lethal autosomal recessive disorder with an estimated incidence of 1 in 355 live births in the Hutterite population. A few cases have been reported in other populations. Here, we report the results of a genome-wide scan and fine mapping of the BCS locus in Hutterite families. By linkage and haplotype analysis the BCS locus was mapped to a 3.5 cM segment (1.9 Mbp) in chromosome region 12p13.3 bounded by F8VWF and D12S397. When genealogical relationships among the families were taken into account in the linkage analysis, the evidence for linkage was stronger and the number of potentially linked regions was reduced to one. Under the assumption that all the Hutterite patients were identical by descent for a disease-causing mutation, haplotype analysis was used to infer likely historical recombinants and thereby narrow the candidate region to a chromosomal segment shared in common by all the affected children. This study also demonstrates that BCS and cerebro-oculo-facial-skeletal syndrome (COFS) are genetically distinct.     

Oral tolerance in protein-deprived mice. I. Profound antibody tolerance but impaired DTH tolerance after antigen feeding.

We have examined the effects of protein deprivation on the induction of oral tolerance for systemic antibody and DTH responses to the protein antigen ovalbumin (OVA). Mice were fed 4% or 24% protein diets from weaning and given a single feed of OVA 2 weeks later (short-term deprivation) or after 10 weeks (long-term deprivation). Tolerance for serum antibody responses was more profound in protein-deprived animals than in 24% protein-fed control groups. Conversely, tolerance for DTH responses was impaired in protein-deprived mice. This was demonstrated both for short-term deprivation, where nutritional rehabilitation after OVA feeding was necessary to demonstrate this effect on DTH, and for long-term deprivation. Furthermore, the effect of short-term deprivation on tolerance for DTH responses was similar to that observed after cyclophosphamide pretreatment of OVA-fed mice. Protein deprivation has disparate effects on the humoral and cell-mediated limbs of oral tolerance, and our results support the hypothesis that this regime selectively depletes a population of suppressor T cells responsible for the fine control of DTH tolerance.     

Isodicentric X chromosome in a moderately tall patient with gonadal dysgenesis: lack of effect of functional centromere on inactivation pattern.

An isodicentric X chromosome (46, X idic (X)(pter leads to qter::qter leads to pter)) with a single functioning centromere was found in all lymphocytes and fibroblasts examined from a female patient 171.5 cm in height presenting with primary amenorrhoea. Replication of the abnormal chromosome was consistently late. In some cells the pattern was asymmetrical but the asymmetry did not appear to relate to the position of the active centromere.     

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.     

Small fiber diameter fibro-porous meshes: tissue response sensitivity to fiber spacing

The purpose of this research was to determine if fiber spacing for small fiber diameter fibro-porous meshes affected tissue response in vivo. Disk-shaped polyurethane meshes, with mean fiber diameters of 7.6 microm and fiber spacing between 6 and 68 microm, were implanted in rat subcutaneous dorsum for 5-week intervals and then prepared for light microscopy and morphological analysis. Results showed that implants with 12- to 68-microm spacing had no histologically apparent fibrous capsule around the perimeter, a result different from that for 6-microm spacing samples that had a capsule around a mean of 34.2% of the perimeter. For the 12- to 68-microm spacing range, a mean of 21.0% of individual fibers within the meshes were encapsulated. Qualitatively, it appeared that larger fibers were encapsulated more frequently than smaller ones. When nodeless or baggy meshes were implanted, cells tended to cluster three or more fibers into groups and then encapsulate each group. Over the 6- to 68-microm spacing range, cell nuclei volume fraction within the meshes increased from the 6- to the 29-microm spacing (p = 0.000) and then decreased from the 29- to the 68-microm spacing (p = 0.015). There was a trend of an increase in local vessel volume fraction with spacing over the 6- to 68-microm range, though the relationship was weak. The results indicate that the reason for the lack of encapsulation of small-fiber fibro-porous meshes is not exclusively a pore boundary explanation, as is proposed for small-pore porous meshes.