Lysis of sensitized sheep erythrocytes in human sera deficient in the second component of complement

Analysis of C-dependent lysis of sensitized SRBC by C2-deficient sera (C2D) led to the characterization of a C2 bypass pathway. Lysis in the total hemolytic C assay by C2D sera was Ca2+-dependent and required a high concentration of hemolysin to sensitize E. Selective component depletion indicated a requirement for C1 and C4 of the classical pathway (CP) and proteins B, P, and probably D of the alternative pathway (AP). Total hemolytic C could be restored to normal in these C2D sera by utilizing heavily sensitized E or by the addition of a supranormal concentration of B. This system most closely resembles a pathway described by J. E. May and M. M. Frank which requires antibody, C1, and the AP but not C4 or C2. It differs in its requirement for C4. We hypothesize that this pathway represents vestiges of a more primitive C pathway. It becomes evident and possibly clinically important in the setting of C2 deficiency, by allowing C activation, other than the AP, and perhaps in normal individuals, by damaging microorganisms that have evolved means to inhibit early components of the CP.     

The precise radioimmunoassay of adenosine: minimization of sample collection artifacts and immunocrossreactivity.

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nM (312.5 fmol) of underivatized adenosine and cross-reacts less than 0.02% with adenine nucleotides and guanosine and not at all with 1 mM inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nM (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-benzyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in anti-sera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2',3'-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO4/Ba(OH)2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel "stop" solution that was optimized to inhibit adenosine formation from AMP by greater than 99% and to inhibit adenosine uptake into red cells and degradation by greater than 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.     

Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.

An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.     

Shyness and boldness in humans and other animals

The shy-bold continuum is a fundamental axis of behavioral variation in humans and at least some other species, but its taxonomic distribution and evolutionary implications are unknown. Models of optimal risk, density- or frequency-dependent selection, and phenotypic plasticity can provide a theoretical framework for understanding shyness and boldness as a product of natural selection. We sketch this framework and review the few empirical studies of shyness and boldness in natural populations. The study of shyness and boldness adds an interesting new dimension to behavioral ecology by focusing on the nature of continuous behavioral variation that exists within the familiar categories of age, sex and size.     

Altruism,tit for tat and ‘outlaw’ genes

Summary In a prior study we combined game theory and inclusive fitness models to examine whether the guarded altruism that can evolve among non-relatives (tit for tat, TFT) might also evolve among close relatives, supplanting unconditional altruism. In most cases, TFT replaced unconditional altruism in family-structured models. Even when TFT is selected at a single locus, however, by withholding altruism from non-reciprocating relatives it may qualify as an outlaw from the standpoint of modifier genes at other loci. Here we examine this possibility with a series of haploid, two-locus models in which a modifier gene transforms TFT into unconditional altruism. The modifier allele spreads to fixation whenever Hamilton's Rule is satisfied, resulting in an unconditional altruist replacing the TFT strategy. As such, TFT may be regarded as an outlaw vulnerable to suppression by alleles at other loci.     

A High-Resolution Interval Map of the q21 Region of the Human X Chromosome

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.     

Mucin-associated T antigens in benign and malignant epithelium of the gall bladder,extrahepatic bile ducts and ampulla of Vater

The mucin-associated antigens Tn, sialosyl-Tn (STn), T and sialosyl-T (STAg) antigens accumulate through aberrant and incomplete glycosylation in malignant epithelial cells. Their diagnostic and prognostic significance in tumours of the colon and cervix has been described, and a possible role for Tn antigen in cell-to-cell adhesion has been suggested. These antigens have been demonstrated through peanut agglutinin (PNA) lectin binding and more recently using specific monoclonal antisera. Differences between the two methods have been described, which may be due to fixation schedules and/or specificity. We have investigated the effect of fixation on the binding of biotinylated PNA lectin and compared its reactivity with the immunoreactivity of monoclonal antisera to Tn, STn, T and STAg antigens in benign and malignant epithelium of the gall bladder, extrahepatic bile ducts and ampulla of Vater. We found that short-term fixation in formol sublimate resulted in poor PNA binding. All other tested fixation schedules showed strong perinuclear binding, similar to that found on cryostat sections. When compared with monoclonal antisera, PNA binding demonstrated the lowest specificity in benign epithelium. In both benign and malignant epithelium, the two methods cannot substitute for each other. STn and STAg antigens were found to be oncodevelopmental throughout the extrahepatic biliary tract. When used in a panel, they are useful as diagnostic markers of malignancy in gall bladder epithelium.     

Plants interact with microbial polysaccharides

Plants are resistant to almost all of the microorganisms with which they come in contact. In response to invasion by a fungus, bacterium, or a virus, many plants produce low molecular weight compounds, phytoalexins, which inhibit the growth of microorganisms. Phytoalexins are produced whether or not the invading microorganism is a pathogen. The production of phytoalexins appears to be a widespread mechanism by which plants attempt to defend themselves against pests. Molecules of microbial origin which trigger phytoalexin accumulation in plants are called elicitors. Structural polysaccharides from the mycelial walls of several fungi elicit phytoalexin accumlation in plants. Approximately 10 ng of the polysaccharide elicits the accumulation in plants of more than sufficient amounts of phytoalexin to stop the growth of microorganisms in vitro. The best characterized elicitors have been demonstrated to be β-1,3-glucans with branches to the 6 position of some of the glucosyl residues. Oligosaccharides, produced by partial acid hydrolysis of the mycelial wall glucans, are exceptionally active elicitors. The smallest oligosaccharide which is still an effective elicitor is composed of about 8 sugar residues. Bacteria also elicit phytoalexin accumulation in plants, but the Rhizobium symbionts of legumes presumably have a mechanism which allows them to avoid either eliciting phytoalexin accumulation or the effects of the phytoalexins if they are accumulated. The lectins of legumes bind to the lipopolysaccharides of their symbiont, but not of their non-symbiont, Rhizobium. It is not known whether the lectin-lipopolysaccharide interaction is involved with the establishment of symbiosis. However, evidence will be presented that suggests that lectins are, in fact, enzymes capable of modifying the structurs of the lipopolysaccharides of their symbiont, but not of their non-symbiont, Rhizobium. It will also be shown that the lipopolysaccharides isolated from different Rhizobium species and from different strains of individual Rhizobium species have different sugar compositions. Thus, the different strains of a single Rhizobium species are as different from one another as the different species of Salmonella and other gram-negative bacteria. This conclusion is substantiated by experiments demonstrating that antibodies to the lipopolysaccharide from a single Rhizobium strain can differentiate that strain from other strains of the same species as well as from other Rhizobium species. The role in symbiosis of the strain-specific O-antigens is unknown.     

The pharmacokinetics of tritiated ethinylestradiol in the rabbit

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life ($\text{t}\text{1}\text{2}$) of 5.5 min and the second, apparently terminal exponential was also rapid ($\text{t}\text{1}\text{2}\text{= 69}\text{min}$). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterohepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administra tion of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.