Channel catfish (Ictalurus punctatus) muscle protein isolate performance processed under different acid and alkali pH values.

Channel catfish (Ictalurus punctatus) muscle was subjected to 6 protein extraction and precipitation techniques using acid solubilization (pH 2.0, 2.5, and 3.0) or alkaline solubilization (pH 10.5, 11.0, 11.5) followed by precipitation at pH 5.5. The catfish protein isolate was compared with ground defatted white muscle. Alkali-processed catfish showed increased gel rigidity, gel strength, and gel flexibility compared to acid-processed catfish, which exhibited inconsistent functional performance, increasing and decreasing gel rigidity, gel strength, and gel flexibility. The gel rigidity (G') at pH 3.0 in the absence of salt had the highest G' of the acid treatments and was not significantly different from the alkaline-treated catfish muscle (P>0.05). However in the presence of added salt pH treatment it had the lowest G' and was different from alkaline treatments (P<0.05) during break force testing. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a potentially broad range of applications. This range of applications is possible due to the modification of the textural properties of catfish muscle protein produced using different acidic or alkaline pH solubility treatments.

Reducing chimera formation during PCR amplification to ensure accurate genotyping.

Measurements of population diversity are fundamental to the reconstruction of the evolutionary and epidemiological history of organisms. Commonly used protocols to measure population diversity using the polymerase chain reaction (PCR) are prone to the introduction of artificial chimeras. These are often difficult to detect and can confound the correct interpretation of results due to the false generation of recombinants when the underlying DNA sample contains multiple distinct templates. This study presents a standardised procedure to suppress the formation of artificial chimeras during PCR amplification. The solution is based on the accurate determination of the efficiency and end point of the log-linear phase of a PCR. This procedure will facilitate the generation of data sets that more accurately reflect the underlying population diversity rather than artifacts introduced by the process itself.

The null oncogene hypothesis and protection from cancer.

Tumour progression involves the inactivation of tumour suppressor genes and the activation of proto-oncogenes. Inactivation of both copies of a tumour suppressor gene is required for carcinogenesis, while germline deletion or inactivation of one copy results in an increase in the risk of cancer and is responsible for many of the known hereditary cancer syndromes. In contrast, activation of only one copy of a proto-oncogene is required for carcinogenesis. Germline deletion or inactivation of one copy of a proto-oncogene halves the risk of activation at this locus. We propose that studies of high risk cancer patients will show such "null oncogene" mutations.

Rapid disease progression and the rate of spread of the HIV epidemic.

Traditional HIV epidemic models suggest that rapid disease progression may lead to reduced transmission because of a decreased time for the spread of the virus. We present a transmission model structured by age and duration of infection, and propose that rapid disease progression may lead to higher viraemia at a younger age, when individuals are more sexually active. We suggest that rapid disease progression should be considered as a factor in the scale of the HIV epidemic in Africa.

HIV-1 variation diminishes CD4 T lymphocyte recognition.

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4(+) T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4(+) T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1(+) patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4(+) T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4(+) T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1(+) patients which fail to stimulate the T cell antigen receptor of HLA class II-restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II-restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.

Use of complete eluted peptide sequence data from HLA-DR and -DQ molecules to predict T cell epitopes, and the influence of the nonbinding terminal regions of ligands in epitope selection.

In diseases with a strong association with an HLA haplotype, identification of relevant T cell epitopes may allow alteration of the pathologic process. In this report we use a reverse immunogenetic approach to predict possible HLA class II-restricted T cell epitopes by using complete pool sequencing data. Data from HLA-DR2(B1*1501), -DR3(B1*0301), -DQ2(A1*0501, B1*0201), and -DQ8(A1*0301, B1*0302) alleles were used by a computer program that searches a candidate protein to predict ligands with a relatively high probability of being processed and presented. This approach successfully identified both known T cell epitopes and eluted single peptides from the parent protein. Furthermore, the program identified ligands from proteins in which the binding motif of the HLA molecule was unable to do so. When the information from the nonbinding N- and C-terminal regions in the pool sequence was removed, the ability to predict several ligands was markedly reduced, particularly for the HLA-DQ alleles. This suggests a possible role for these regions in determining ligands for HLA class II molecules. Thus, the use of complete eluted peptide sequence data offers a powerful approach to the prediction of HLA-DQ and -DR peptide ligands and T cell epitopes.

HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

Analysis of peptide-binding motifs for two disease associated HLA-DR13 alleles using an M13 phage display library.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate 'sequence motif' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.

Reverse immunogenetics: from HLA-disease associations to vaccine candidates.

Analysis of the human leukocyte antigens (HLA) in patients with either infectious or autoimmune diseases has led to the identification of several HLA alleles associated with either resistance or susceptibility to disease. Understanding the role of HLA molecules in the presentation of peptide antigens to T cells has led to the use of 'reverse immunogenetics': a novel approach to analysing the key antigenic peptides that are presented by the relevant HLA molecules. Recent advances in the analysis of naturally occurring peptides bound to HLA molecules has allowed the direct identification of antigenic peptides from living cells and has supported the development of vaccine candidates, such as the liver-stage antigen 1 in malaria.

Antagonists or altruists: do viral mutants modulate T-cell responses?

The ability of altered peptide ligands (APLs) to modify or downregulate T-cell responses in vitro has generated much interest. Recently, it was proposed that viral variants may contain mutations in cytotoxic T lymphocyte (CTL) epitopes that allow mutual antagonism, and that these combine to downregulate the host immune response. Here, Miles Davenport discusses some inherent problems with this simple model and proposes a closer evaluation of the effects of viral competition on host-viral interactions.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

An empirical method for the prediction of T-cell epitopes.

Identification of T-cell epitopes from foreign proteins is the current focus of much research. Methods using simple two or three position motifs have proved useful in epitope prediction for major histocompatibility complex (MHC) class I, but to date not for MHC class II molecules. We utilized data from pool sequence analysis of peptides eluted from two HLA-DR13 alleles to construct a computer algorithm for predicting the probability that a given sequence will be naturally processed and presented on these alleles. We assessed the ability of this method to predict known self-peptides from these DR-13 alleles, DRB1(*)1301 and *1302, as well as an immunodominant T-cell epitope. We also compared the predictions of this scoring procedure with the measured binding affinities of a panel of overlapping peptides from hepatitis B virus surface antigen. We concluded that this method may have wide application for the prediction of T-cell epitopes for both MHC class I and class II molecules.

Prevalence of cystine and urate uroliths in bulldogs and urate uroliths in dalmatians.

Data were evaluated from all dogs admitted to the University of Minnesota Veterinary Teaching Hospital (UMVTH) between June 1, 1981 and Dec 31, 1991. During this period, uroliths were retrieved and analyzed from 452 of 37,574 dogs admitted. The odds that uroliths from Bulldogs were composed of cystine were 32.3 times greater than for other breeds evaluated. The odds that a Bulldog admitted was affected with cystine uroliths were 154.1 times greater than for other breeds. Cystine uroliths were retrieved only from male Bulldogs. The odds that uroliths from Bulldogs were composed of urate were 7.9 times greater than for other breeds. The odds that a Bulldog admitted was affected with urate uroliths were 43.0 times greater than for other breeds. Male Bulldogs were 14.3 times more likely to be affected with urate uroliths than were females. The odds that uroliths from Dalmatians were composed of urate were 228.9 times greater than for other breeds. The odds that a Dalmation admitted was affected with urate uroliths were 122.4 times greater than for other breeds. Male Dalmatians were 16.4 times more likely to be affected with urate uroliths than were females. Data also were evaluated from files of canine uroliths submitted to the Minnesota Urolith Center for quantitative mineral analysis between June 1, 1981 and Dec 31, 1991. During this period, 94 of 11,188 uroliths analyzed were obtained from Bulldogs and 387 were obtained from Dalmatians. The odds that uroliths retrieved from Bulldogs were composed of cystine were 40.7 times greater than for other breeds. Cystine uroliths were retrieved only from male Bulldogs.(ABSTRACT TRUNCATED AT 250 WORDS)