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K L Knight

Publications and source records attributed to K L Knight.

At least 73 records · Page 4Linked to original sources
PubMed1994

Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli.

Assembly of RecA subunits into long, helical oligomers is required for its roles in recombinational DNA repair and homologous genetic recombination. The crystal structure of RecA reveals an extensive network of amino acid residues that lie at the subunit boundaries. We have introduced a large set of substitutions at 5 clustered residues, which are shown in the crystal structure to make specific contacts with positions in the neighboring monomer. We find that 3 of the 5 residues are important for RecA function (Lys216, Phe217, and Arg222), whereas the other 2 (Asn213 and Tyr218) are not. The patterns of functionally allowed substitutions provide insight into the chemical and steric constraints required at these positions.

Amino Acid Sequence↗
PubMed1994

Neonatal VH, D, and JH gene usage in rabbit B lineage cells.

The neonatal antibody repertoire in both mouse and humans differs from that of the adult repertoire in that the neonatal repertoire uses a limited set of JH-proximal VH genes but the adult repertoires use many different VH genes. Rabbits are unusual in that adults use only three or four VH genes, with approximately 80% of the B cells using VH1, the 3'-most VH gene. To investigate whether the repertoire of neonatal rabbits differs from that of adults, we analyzed VH, D, and JH gene usage in B cells of neonatal rabbits. A total of 68 rearranged VDJ genes was cloned from mRNA and genomic DNA isolated from lymphoid tissues of newborn to 10-day-old rabbits. We found that 74% of the VDJ gene rearrangements utilized VH1 and 15% utilized the genes that we designated VHx or VHy. From the remaining VDJ genes we identified seven novel VH genes, one, VHz, which was found in mRNA. We conclude that the repertoire of utilized VH genes in neonates is limited and is similar to that of adult rabbits. We also found the D1, D2a, D2b, and JH4 gene segments preferentially rearranged. We suggest that the preferential usage of VH, D, and JH gene segments in VDJ genes is caused by preferential rearrangement rather than by selective expansion of B cells that utilize the gene segments.

Amino Acid Sequence↗
PubMed1994

Generating the antibody repertoire in rabbit.

We describe a model for B cell development and generation of the antibody repertoire in rabbits. In this model, B cells develop early in ontogeny, migrate to GALT, and undergo the first round of diversification by a somatic gene conversion-like process and by somatic mutation. We designate the repertoire developed by this mechanism as the primary antibody repertoire and it is this repertoire that makes the rabbit immunocompetent. We invoke GALT as the site for development of the primary repertoire because (1) surgical removal of GALT from neonatal rabbits results in highly immunocompromised animals, (2) in germfree rabbits essentially no lymphoid development occurs in GALT and the rabbits are immunoincompetent, and (3) the follicular development of rabbit GALT is highly similar to that of the chicken bursa, the site in which the primary antibody repertoire develops by somatic gene conversion in chicken. We suggest that once the primary antibody repertoire is formed, it is maintained by self-renewing CD5+ B cells and is expanded to a secondary antibody repertoire after the B cells encounter antigen.

Amino Acid Sequence↗
PubMed1994

Lymphoid and non-lymphoid tumors in E kappa-myc transgenic rabbits.

We developed transgenic rabbits with a DNA construct containing the proto-oncogene c-myc conjugated to the Ig kappa-chain enhancer gene, E kappa. One of four transgenic rabbits was mated to a normal rabbit and we used the offspring to develop a colony of rabbits carrying the E kappa-myc transgene in their germline. Of a total of 19 E kappa-myc transgenic rabbits, eight developed tumors. The tumors were characterized histologically and four were diagnosed as lymphoma, and one each was diagnosed as embryonic carcinoma, hepatoma, ovarian carcinoma and basal cell carcinoma. By Southern analysis, we showed the four lymphomas were of B-lymphoid lineage and by nucleotide sequence analysis we found three of them most likely used VH1 in their VDJ gene rearrangements. Cells from the embryonic carcinoma, the hepatoma and two of the B-lymphomas were adapted to tissue culture. We discuss the possibility that tumors of non-lymphoid origin develop in the E kappa-myc transgenic rabbits because of the potential for NF-kappa B to activate the kappa-enhancer in cells other than B-lymphoid lineage cells.

Animals↗
PubMed1993

Mutagenesis of the P-loop motif in the ATP binding site of the RecA protein from Escherichia coli.

Using a combinatorial cassette mutagenesis procedure we have introduced a number of mutations into 10 codons that define the P-loop motif within the ATP binding site of the Escherichia coli RecA protein. The recombinational proficiency of the recA mutants was determined using three genetic assays: survival in the presence of 4-nitroquinoline-1-oxide, survival following UV irradiation and the ability to support plaque formation by a red-gam-Chi+ lambda phage. While no amino acid substitutions were allowed at the four residues that define the P-loop consensus sequence, a variety of changes at the other positions in this region were observed that allowed full or partial RecA function. This occurred despite the fact that these residues are very highly conserved among 22 eubacterial RecA proteins, and represent the most conserved stretch of 10 contiguous residues in the entire RecA sequence. Our results show that these residues display marked differences in the ability to support mutations. The mutability of each of these 10 residues is discussed in terms of possible functional and/or structural roles.

4-Nitroquinoline-1-oxide↗
PubMed1993

Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues.

Molecular cloning techniques have recently demonstrated that rabbit has 13 different IgA C alpha H chain genes. This is in contrast to human and mouse that have only two and one C alpha heavy chain genes, respectively. In previous studies, nucleotide sequence analysis indicated that the 13 rabbit C alpha genes were potentially functional, and in vitro expression experiments showed that at least 12 of these genes were expressible. To understand the role of these multiple IgA isotypes we analyzed RNA of various lymphoid tissues for the presence of mRNA representing each of the multiple C alpha genes. We used the RNase protection assay with probes that are specific for the 13 different C alpha genes and we consistently found that at least 10 of the C alpha genes are expressed, albeit at different levels, in gut (small intestines), appendix, mesenteric lymph node, and mammary tissue. However, in salivary gland (submandibular), only seven of these genes are expressed at significant levels and in lung and tonsil only one C alpha gene, C alpha 4, is expressed at a level comparable to its expression in other tissues. Analysis of RNA of Peyer's patch showed differences in the level of C alpha gene expression between different animals and between different Peyer's patches of the same rabbit; in some cases, most of the C alpha genes were expressed, but in some cases only C alpha 4 was expressed at a significant level. Inasmuch as C alpha 4 is the 5' most C alpha gene we propose that IgA-producing cells are derived from B cells that have initially undergone isotype switching to C alpha 4 and we discuss various mechanisms that could explain switching to the more 3' C alpha genes.

Animals↗
PubMed1993

The effects of ice and compression wraps on intramuscular temperatures at various depths.

While ice and compression wraps are commonly used to treat musculoskeletal injuries, the literature describing intramuscular temperatures has not addressed the combination of ice and compression wraps. The purpose of this study was to evaluate intramuscular temperatures at three sites on the anterior thigh (skin surface, 1 cm below the fat layer, and 2 cm below the fat layer) using both ice and compression wraps. Temperatures were recorded in 11 subjects with an isothermex, using implantable and surface thermocouples. Each subject was tested under four conditions: control, compression only, ice only, and ice + compression according to a balanced Latin square. Surface and intramuscular temperatures were recorded at 30 second intervals during 5 minutes of preapplication, 30 minutes application, and 20 minutes postapplication. A repeated measures ANOVA and Duncan post hoc tests were used to evaluate peak temperature differences between the treatment conditions and the depths of measurement. Both ice alone and ice + compression produced significant cooling at all three depths (F(6,60) = 168.5, p<.0005). Likewise, during the 20-minute postapplication period, these temperatures did not return to their preapplication levels. The compression-only condition produced significant warming at the skin surface, but did not have any effect on intramuscular temperature. At all depths, the ice + compression condition produced significantly cooler temperatures than ice alone. We suggest that compression increases the effectiveness of ice in reducing tissue temperatures. Therefore, ice combined with compression should be more effective than ice alone in reducing the metabolism of injured tissue. This provides an additional rationale for combining ice with compression in treating acute musculoskeletal injuries.

Journal Article↗
PubMed1992

CD5+ B cells predominate in peripheral tissues of rabbit.

Restricted usage of VH genes is observed in rabbit B lymphocytes and in human and murine CD5 B lymphocytes. This observation raised the possibility that most rabbit B lymphocytes were CD5+. To investigate this we cloned the CD5 gene from a rabbit cosmid library, using a probe derived from human CD5 cDNA. The rabbit CD5 gene was transfected into a murine T cell line and then we used the transfectants to develop anti-rabbit CD5 mAb. By Western blot analysis, the mAb reacted with a 67-kDa protein in lysates prepared from mesenteric lymph node and spleen cells. We determined the frequency of CD5+ B lymphocytes in peripheral lymphoid tissues of adult rabbits by two-color immunofluorescence analysis using anti-CD5 mAb and anti-L chain antibodies. The analysis showed that essentially all peripheral B lymphocytes in adult rabbits express CD5. The observation that CD5 is expressed on nearly all rabbit B lymphocytes contrasts markedly to mouse and human, where only a small number of B lymphocytes express CD5. We propose that most peripheral B lymphocytes in rabbit, as in chicken, develop early in ontogeny and are maintained throughout life by a self-renewing process.

Animals↗
PubMed1992

Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor.

Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition. Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions. The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins. In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators. Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14. The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator.

Base Sequence↗
PubMed1992

Limited repertoire of utilized VH gene segments in a VHa3-allotype-suppressed rabbit.

Between 70 and 90% of serum Ig molecules of normal laboratory rabbits bear one of the serologically defined VHa allotypic specificities, a1, a2, or a3, and are termed VHa+ (a-positive) molecules; the remaining 10-30% of Ig molecules that do not have VHa allotypic specificities are designated VHa- (a-negative). The repertoire of utilized VHa(+)-encoding gene segments has been examined extensively, but only limited studies of the repertoire of utilized VHa(-)-encoding gene segments in normal rabbits have been done. To examine the repertoire of utilized VHa- gene segments, we analyzed VH-encoding cDNA clones from mRNA of a VHa-allotype-suppressed rabbit whose serum Ig was primarily VHa-. For VHa suppression a newborn a3/a3 rabbit was injected periodically with anti-VHa3 antiserum; when it was 2 months of age and its serum Ig was greater than 94% VHa-, cDNA clones were generated from splenic RNA. The nucleotide sequences of eight putative VHa(-)-encoding cDNA clones were compared to those of eight cDNA clones generated from RNA of non-suppressed a3/a3 rabbits. The presumed VHa3-encoding cDNA clones from the non-suppressed rabbits appeared to derive from VH1-a3, the 3'-most germline VH gene segment. In contrast, the VHa(-)-encoding cDNA clones from the suppressed rabbit were distinctly different from VH1 and were most probably derived from germline VH segments other than VH1. Because the expressed VHa- gene repertoire was highly restricted, we propose that VHa- molecules in a3/a3 rabbits may be derived from as few as three germline VH gene segments.

Amino Acid Sequence↗
PubMed1992

Restricted VH gene usage and generation of antibody diversity in rabbit.

The presence of VHa allotypic specificities on nearly all rabbit Ig molecules has perplexed immunologists for many years. How could these allotypic specificities be inherited as if controlled by alleles if the germline has hundreds of VHa allotype-encoding genes and if most of these genes are used in VDJ gene rearrangements. I review recent data indicating that the allelic inheritance of the VHa allotypes can be explained by preferential utilization of the D-proximal VH gene VH1 in VDJ gene rearrangements. The preferential usage of one VH gene, however, limits the contribution of combinatorial joining of multiple VH, D and JH gene segments to the generation of antibody diversity. The roles of somatic gene conversion and somatic mutation in generating antibody diversity are discussed. Further, the limited usage of germline VH genes in normal, allotype-suppressed and the mutant Alicia rabbit as well as the molecular basis of latent allotypes and VH/CH recombinants is reviewed.

Animals↗
PubMed1992

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Journal Article↗
PubMed1992

Habituation to cold-pain during repeated cryokinetic sessions.

Clinicians claim that patients habituate to cold-induced pain during cryokinetic treatments, but this has not heretofore been tested. We treated the right ankle of 38 subjects with a simulated cryokinetic treatment daily for 8 days using either 1 degrees or 5 degrees C water. On days 9 and 10, the right ankle was treated with the opposite temperature and the opposite ankle (left) was treated with the habituation temperature. Cold-induced pain was recorded five times each day (after each ice immersion bout) using Borg's Perceived Pain Scale and the McGill Pain Questionnaire. There was a sharp decrease in pain from days 1 through 5, but no difference between days 5 through 8. Pain during bout one was significantly greater than the following four bouts for all days except day 1. Location of pain changed between days but not between bouts. The instep was the most frequent location of pain for the first 3 days. The choice of "no specific location" increased steadily from day 2 to 8. The number of descriptor terms chosen on the McGill Pain Questionnaire decreased from day 1 to day 8. Exceptions to this were the terms cool, cold, freezing, and numb. Common terms chosen on days 1 through 3 were throbbing, sharp, burning, tingling, hurting, and nagging. On days 9 and 10, pain in the opposite (left) limb was greater than pain at the end of right limb habituation, but similar to day 1 of habituation. Right limb immersion with a lower temperature resulted in greater pain than that perceived on day 8. Daily repeated cryokinetic treatments are sufficient to produce habituation to cold-induced pain. Habituation was specific to the limb treated and temperature of habituation; thus, we conclude it is primarily physiological with some psychological influence. Athletic trainers are justified in telling patients who are undergoing ice water immersion that the cold pain will diminish with repeated applications.

Journal Article↗
PubMed1992

Sensory perception of the foot and ankle following therapeutic applications of heat and cold.

Many athletes are treated with hot and cold modalities prior to therapeutic exercise, but the effects of these treatments on sensory perception are not clear. The purpose of this study was to examine the effects of hot and cold treatments on sensory perception. We recruited 21 volunteer subjects, who reported for testing on three separate occasions. One of three treatments was applied to the left ankle and foot each day for 20 minutes: cold immersion, hot immersion, or quiet sitting (control). Three variables were measured following treatment: topagnosis, two-point discrimination, and one-legged balance. We assigned treatments and the testing order according to a Greco Latin square. Data were analyzed using a multivariate analysis of variance (MANOVA). No significant differences were detected for the three dependent measures, suggesting that therapeutic applications of heat and cold do not affect sensory perception. These findings indicate that heat and cold applications can be used prior to therapeutic exercise programs without interfering with normal sensory perception as do other analgesic and anesthetic agents. For example, the hypalgesic effect of cold, which is essential to cryokinetics, can be realized without fear of altered sensory perception.

Journal Article↗