The major histocompatibility class II alpha chain in salmonid fishes

In this study the characterisation of the Atlantic salmon (MhcSasa-DAA) and rainbow trout (MhcOnmy-DAA) class II alpha chain cDNA sequences is presented. The DAA sequences from these two salmonid species showed a high degree of similarity, although the Onmy-DAA^*03 cDNA sequence differed in the cytoplasmic region. Interestingly, the Onmy-DAA^*02 sequence has lost the second cysteine in the alpha-1 domain. However, another cysteine is present in this sequence 7 positions downstream of the cysteine which is substituted for a leucine. Despite a thorough search, only a single locus of expressed class II alpha chain sequences was identified in both salmonid species. Amplification by PCR and sequencing of the alpha-1 domain from genomic DNA of three Atlantic salmon, identified four different variants assumed to have derived from this single locus. Two of these variants originated from one individual and are likely functional alleles.     

Comparative genome analysis of the primary sex-determining locus in salmonid fishes

We compared the Y-chromosome linkage maps for four salmonid species (Arctic charr, Salvelinus alpinus; Atlantic salmon, Salmo salar; brown trout, Salmo trutta; and rainbow trout, Oncorhynchus mykiss) and a putative Y-linked marker from lake trout (Salvelinus namaycush). These species represent the three major genera within the subfamily Salmoninae of the Salmonidae. The data clearly demonstrate that different Y-chromosomes have evolved in each of the species. Arrangements of markers proximal to the sex-determining locus are preserved on homologous, but different, autosomal linkage groups across the four species studied in detail. This indicates that a small region of DNA has been involved in the rearrangement of the sex-determining region. Placement of the sex-determining region appears telomeric in brown trout, Atlantic salmon, and Arctic charr, whereas an intercalary location for SEX may exist in rainbow trout. Three hypotheses are proposed to account for the relocation: translocation of a small chromosome arm; transposition of the sex-determining gene; or differential activation of a primary sex-determining gene region among the species.     

Conformational properties of striated muscle tropomyosins from some salmonid fishes

The conformational stability of tropomyosins from salmonids fishes has been investigated under a variety of conditions (salt, pH and osmolyte) using electronic circular dichroism. Every salmonid tropomyosin (from: fast skeletal muscle; slow skeletal muscle and heart) is less resistant to heat-induced denaturation than rabbit alpha-striated tropomyosin. Induction of unfolding, by application of a linear temperature gradient, yields a distinct profile for each protein (0.1 M salt, pH 7, plus dithiothreitol): fast tropomyosin (Tms 24 and 40°C major); cardiac tropomyosin (Tm, 36°C) and slow tropomyosin (Tms, 39 major and 47°C). Correlation of these results, and others obtained under different solvent conditions, with the known sequences (Jackman DM, Waddleton DM, Younghusband B, Heeley DH (1996) Further characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish. Eur. J. Biochem. 242:363–371) provides insight into how the coiled-coil may have adapted to cold. The most variable sections of sequence encompass residues 9–49, 73–87 and 172–216. In two of these regions there is a pair of closely-spaced glycines, namely at residues 24 and 27 in fast skeletal tropomyosin and residues 83 and 87 in cardiac tropomyosin. A region of low stability is located in the carboxy-terminal half of the isoform from fast skeletal muscle. This segment cooperatively unfolds in the 20° range and accounts for 20% of the total far-UV ellipticity change under reducing conditions. It is unresponsive to increasing ionic strength and the presence of osmolyte but is sensitive to oxidation at cysteine 190. A likely contributor to the relative instability is a substitution at position 179 whereby fast skeletal tropomyosin, but not the other tropomyosins under examination, has lost a “d” position alanine in the fifth cluster and gained a polar side-chain.     

Morphology,distribution, and specificity of olfactory receptor cells in salmonid fishes

Electrophysiological and anatomical investigations of the olfactory epithelium of three salmonid species have been coordinated. Different corridors in the olfactory rosettes were examined by recording of the electro-olfactogram (EOG) using two recording electrodes simultaneously against one common indifferent electrode. Biopsies comprising the two lamellae bounding each of the examined corridors were examined by scanning electron microscopy (SEM). The electrophysiological results confirm a higher relative density of bile salt-receptors in the peripheral parts of all the corridors, and a higher relative density of amino acid-receptors in central parts. The SEM micrographs show that ciliated receptor cells are more numerous than microvillous ones. The density of ciliated receptor cells increases towards the peripheral margin of each lamella. The density of microvillous receptor cells increases towards the central margin. The correspondence between the electrophysiological and the anatomical observations support the hypothesis that the ciliated receptor cells are those specific to bile salt-like substances, the microvillous those specific to amino acids.     

Functional morphology and biomechanics of the tongue-bite apparatus in salmonid and osteoglossomorph fishes

The tongue-bite apparatus and its associated musculoskeletal elements of the pectoral girdle and neurocranium form the structural basis of raking, a unique prey-processing behaviour in salmonid and osteoglossomorph fishes. Using a quantitative approach, the functional osteology and myology of this system were compared between representatives of each lineage, i.e. the salmonid Salvelinus fontinalis ( N  = 10) and the osteoglossomorph Chitala ornata ( N  = 8). Divergence was found in the morphology of the novel cleithrobranchial ligament, which potentially relates to kinematic differences between the raking lineage representatives. Salvelinus had greater anatomical cross-sectional areas of the epaxial, hypaxial and protractor hyoideus muscles, whereas Chitala had greater sternohyoideus and adductor mandibulae mass. Two osteology-based biomechanical models (a third-order lever for neurocranial elevation and a modified four-bar linkage for hyoid retraction) showed divergent force/velocity priorities in the study taxa. Salvelinus maximizes both force (via powerful cranial muscles) and velocity (through mechanical amplification) during raking. In contrast, Chitala has relatively low muscle force but more efficient force transmission through both mechanisms compared with Salvelinus . It remains unclear if and how behavioural modulation and specializations in the post-cranial anatomy may affect the force/velocity trade-offs in Chitala . Further studies of tongue-bite apparatus morphology and biomechanics in a broader species range may help to clarify the role that osteology and myology play in the evolution of behavioural diversity.     

The immunopathological significance of the heterogeneity of antibody affinity

Mice of two inbred strains, prone to nephritis following neonatal lymphocytic choriomeningitis virus infection, produced antibody of lower affinity to two soluble protein antigens, human serum albumin and transferrin, than did two nephritis-resistant strains. Antigen in adjuvant elicited higher affinity antibody than did antigen in saline, in the nephritis-prone mice. The significance of these findings in the pathogenesis and treatment of nephritis, arthritis and other possible soluble complex disease is discussed.     

The use of monoclonal antibodies in demonstrating the effect of antibody heterogeneity on immune complex size

The effect of antibody heterogeneity on immune complex size was investigated by using monoclonal antibodies. Five monoclonal antibodies which bind different antigenic determinants of human serum albumin were used to produce immune complexes. When one or two different monoclonal antibodies were used, under antigen-excess, antigen-antibody equivalent, and antibody-excess conditions, hardly any immune complexes larger than mouse IgM were produced. On the other hand, in a representative experiment, when five different monoclonal antibodies were used, immune complexes larger than mouse IgM comprised 21.5% of the total antigen under antigen-antibody equivalent conditions, 18.2% under antibody-excess conditions, and 4.3% under antigen-excess conditions. These results indicate that antibody heterogeneity is one of the important factors determining immune complex size.     

The heterogeneity of antibody affinity in inbred mice and its possible immunopathologic significance

The range of relative affinity of antibody to HSA in mice from ten strains is presented. Previously reported high and low affinity strains are not exceptional.

Age, sex, dose and time after immunization have little effect on relative affinity of antibody to HSA and HST.

Antibodies, raised by injecting DNP-HSA or DNP-RSA, and tested by ³H-εDNP-L-lysine, show similar interstrain differences of affinity. This excludes recognition of only a limited proportion of the diverse antigenic sites on complex protein antigens as the sole explanation of this phenomenon.

     

The impact of breathing motion versus heterogeneity effects in lung cancer treatment planning

The purpose of this study is to investigate the effects of tissue heterogeneity and breathing-induced motion/deformation on conformal treatment planning for pulmonary tumors and to compare the magnitude and the clinical importance of changes induced by these effects. Treatment planning scans were acquired at normal exhale/inhale breathing states for fifteen patients. The internal target volume (ITV) was defined as the union of exhale and inhale gross tumor volumes uniformly expanded by 5 mm. Anterior/posterior opposed beams (AP/PA) and three-dimensional (3D)-conformal plans were designed using the unit-density exhale ("static") dataset. These plans were further used to calculate (a) density-corrected ("heterogeneous") static dose and (b) heterogeneous cumulative dose, including breathing deformations. The DPM Monte Carlo code was used for dose computations. For larger than coin-sized tumors, relative to unit-density plans, tumor and lung doses increased in the heterogeneity-corrected plans. In comparing cumulative and static plans, larger normal tissue complication probability changes were observed for tumors with larger motion amplitudes and uncompensated breathing-induced hot/cold spots in lung. Accounting for tissue heterogeneity resulted in average increases of 9% and 7% in mean lung dose (MLD) for the 6 MV and 15 MV photon beams, respectively. Breathing-induced effects resulted in approximately 1% and 2% average decreases in MLD from the static value, for the 6 and 15 MV photon beams, respectively. The magnitude of these effects was not found to correlate with the treatment plan technique, i.e., AP/PA versus 3D-CRT. Given a properly designed ITV, tissue heterogeneity effects are likely to have a larger clinical significance on tumor and normal lung treatment evaluation metrics than four-dimensional respiratory-induced changes.     

Functional heterogeneity of antibody to penicilloyl coated erythrocytes

Human sera with anti-BPO (benzyl penicilloyl) specificity usually fail to lyse erythrocytes coated with BPO groups in vitro, when exposed to either guinea-pig or human C (complement). Of 350 haemagglutinating sera only seven caused lysis at levels 64–128-fold less than the corresponding haemagglutinin titres. However, of 100 sera which failed to cause lysis in vitro, twenty-one appeared capable of deviating part of the complement sequence as measured by assay of residual haemolytic C activity. Both BPO coated cells and alkaline PG solutions were effective antigens, the latter possibly as a result of polymerization which occurs readily in PG (benzyl penicillin) solutions.

Rabbit anti-human IgG sera caused haemolysis of BPO coated cells with adsorbed IgG antibody molecules. In these circumstances haemolysis was most marked with 2-mercaptoethanol reduced sera. Haemagglutination by IgM antibody in non-mercaptoethanol treated sera prevented haemolysis with anti-IgG and C, and this effect appeared to be related to the degree of aggregate formation, possibly by a steric hindrance effect of compact aggregates.

     

Genetic heterogeneity in primary hyperoxaluria type 1: impact on diagnosis

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease characterized by progressive kidney failure due to renal deposition of calcium oxalate. The disease is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. When AGT is absent, glyoxylate is converted to oxalate which forms insoluble calcium salts that accumulate in the kidney and other organs. In the most common phenotype there is a unique phenomenon wherein AGT is mis-targeted to the mitochondria instead of the peroxisomes. The diagnosis of PH1 is complicated by heterogeneity of clinical presentation, course of the disease, biochemical markers, AGT enzymatic activity and genotype. More than 50 mutations and polymorphisms have been reported in the AGT gene; three common mutations accounting for almost 50% of PH1 alleles. The mutations are of all types, with missense making up the largest fraction. There are some mutations with apparent ethnic associations and at least one that appears to be pan-ethnic. Although correlations can in some cases be made between biochemical phenotype and genotype, correlation with clinical phenotype is complicated by the involvement of other genetic and non-genetic factors that affect disease severity. A number of polymorphisms have been described in the AGT gene some of which cause missense changes and, in some cases, alter enzyme activity. As DNA testing becomes more commonly used for diagnosis it is important to correlate observed sequence changes with previously documented changes as an aid to assessing their potential significance.     

Differential susceptibility of salmonid fishes to furunculosis correlates with differential serum enhancement of Aeromonas salmonicida extracellular protease activity

Rainbow trout, Atlantic salmon and brown trout, respectively, exhibit an increasing order of susceptibility to furunculosis. We investigated if this may be due to a differential sensitivity to exotoxins or ability to inhibit the exoprotease which is required by the bacterium to digest protein for growth. Differences were slight in susceptibility to exotoxins and serum inhibition of exoprotease at high exotoxin:serum ratios. However, at low ratios brown trout serum markedly enhanced protease activity, salmon serum had little effect and rainbow trout serum maintained 50% inhibition. This implies that during the early stages of infection the potential for growth of the bacterium would be highest in brown trout and lowest in rainbow trout. Determinations of growth rates in fresh serum support this hypothesis. The enhancement of exoprotease activity by serum of susceptible species may represent an important virulence mechanism.     

The Gardner syndrome: increased tetraploidy in cultured skin fibroblast.

Tetraploidy was increased in skin fibroblast cultures grown in the laboratory at the same time under the same conditions and derived from 2 probands with the Gardner syndrome and 9 affected members of one family as compared to that occurring in cultures from 5 relatives by marriage and 10 normals. Tetraploidy was present at the first subculture (2 weeks after the initial biopsy was cultured), and for each line studied the percentage of dividing cells showing tetraploidy remained constant. The relation of the observed tetraploidy to the increased risk of such patients to develop abnormal growths and cancer has not been established. The increased tetraploidy should be of value in identifying the presence of the gene for the Gardner syndrome in high risk families.     

Identification of multiple sources of charge heterogeneity in a recombinant antibody

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.     

Functional and anatomic heterogeneity in the liver acinus: impact on transport

The site of solute exchange between blood and hepatocytes is the liver acinus, the structural and functional unit of hepatic parenchyma. Because the perfusion of acinar hepatocytes occurs in a sequential manner, differences in solute concentration between hepatocytes located at the inlet (zone 1) and at the outlet (zone 3) of the acinus are predictable. However, the distribution and transport of solutes by hepatocytes of each acinar zone also seem to be influenced by factors other than the sequential perfusion of blood. The concentration of receptors in hepatocytes at each zone, the presence of specific chemical groups on the incoming molecules, and the binding of the solutes to plasma protein are all factors that modify the interaction with, and the cellular concentration attained by, solutes in each acinar zone. Therefore, solute concentration in hepatocytes of each acinar zone may produce a profile of decreasing solute concentration from cells in zone 1 to those in zone 3, a reverse profile from zone 3 to zone 1, or a similar concentration in all hepatocytes.     

The distribution of ancestral secondary cases in Parkinson's disease

Recent evidence suggests a genetic mode of transmission of Parkinson's disease. A multifactorial mechanism is likely, but autosomal dominance with reduced penetrance has not been excluded.
The distribution of ancestral second-degree relatives with Parkinson's disease was evaluated in 12 families. Affected relatives were bilaterally distributed more often than would be expected for autosomal dominance.
The distribution of ancestral secondary cases suggests a multifactorial etiology for most cases of Parkinson's disease. The possibility of dominant gene inheritance in some families is not entirely ruled out.