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Biomedical subjects

K L Knight

Publications and source records attributed to K L Knight.

At least 91 records · Page 5Linked to original sources
PubMed1992

Effects of Prior Physical Activity on Skin Surface Temperature Response of the Ankle During and After a 30-minute Ice Pack Application.

The effects of various durations of treadmill running on ankle skin temperature response during and following ice application were investigated. We measured the ankle skin surface temperature of 12 male subjects with a telethermister (YSI Model 44) during each of three conditions: control and two exercise conditions that involved treadmill running for 15 or 30 minutes, followed by a 30-minute ice pack application and 90 minutes rewarming. The control condition involved no exercise prior to ice pack application. Ankle skin temperature increased significantly during 15 and 30 minutes of exercise, although the temperature difference between the two conditions was not statistically significant. Mean skin cooling temperatures were slightly, though not significantly, higher following exercise than following no exercise. The rate of cooling, however, was unaffected by prior exercise. Mean skin temperatures during rewarming were significantly higher following the exercise conditions, but the rate of rewarming was unchanged by exercise. Mean rewarming temperatures were higher in the 30-minute exercise condition than in the 15-minute exercise condition. Longer ice applications or shorter reapplications may be necessary following exercise of at least 15 minutes, but further investigation is necessary to substantiate this supposition.

Journal Article↗
PubMed1991

VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions.

In this study we investigate the molecular genetic basis for VHa- Ig. Knowing that the expression of VHa allotype Ig is suppressed by neonatal injection of rabbits with anti-VHa allotype antibody, and that the decreased level of VHa allotype Ig, VHa+, in the suppressed rabbits is compensated for by an increase in VHa- Ig, we determined the nucleotide sequences of 41 VDJ genes from a2/a2 rabbits neonatally suppressed for the expression of a2 Ig. We compared these nucleotide sequences to each other and identified two groups of VH sequences. We predict that the molecules of each group are encoded by one germline VH gene. Inasmuch as VHa+ Ig is encoded predominantly by one germline VH gene, VH1, it appears that more than 95% of the VDJ repertoire of rabbits may be encoded by as few as three germline VH genes. A genomic VDJ gene whose VH sequence was similar to those of group I molecules was expressed in vitro and was shown by ELISA to encode molecules of the VHa- allotype, y33. Analysis of the D regions in the VDJ gene indicated that germline D2b and D3 gene segments were preferentially used in the VDJ gene rearrangement. A comparison of sequences of D regions of the 41 VDJ gene rearrangements in 3-, 6-, and 9-wk-old rabbits to sequences of germline D gene segments showed an accumulation of mutations in the D region. Inasmuch as we have previously shown that V regions of rabbit VDJ genes are diversified, in part, by somatic gene conversion, it appears now that rabbit VDJ genes diversify by a combination of somatic mutation and somatic gene conversion.

Amino Acid Sequence↗
PubMed1991

Restricted utilization of germ-line VH genes in rabbits: implications for inheritance of VH allotypes and generation of antibody diversity.

The presence of inherited VH region allotypic specificities, a1, a2 or a3, on nearly all rabbit immunoglobulins has presented a paradox. We know the germline contains hundreds of VH genes, and if we assume that most of these are used in the generation of antibody diversity, then we must ask how have the a allotype-encoding regions been maintained over time? On the other hand, if we assume that only one (or a small number) of these VH gene(s) is (are) used in VDJ gene rearrangements, then, how is antibody diversity generated? To address these questions, we have cloned and determined the nucleotide sequence of the 3'-most germline VH genes from the a1, a2 and a3 chromosomes and shown in each case that the 3'-most H gene, VH1-a1, VH1-a2, or VH1-a3, encodes an a1, a2 or a3 VH region, respectively. Analysis of rearranged VDJ genes from leukemic B cells showed that VH1 was utilized in these rearrangements. Based on these data, we propose that the allelic inheritance of the VH allotypes is explained by the preferential usage of the VH1 gene in VDJ rearrangements. Support for this hypothesis was obtained from analysis of the mutant rabbit Alicia in which most serum Ig molecules do not have VHa allotypic specificities, but instead have so-called VHa-negative Ig molecules. In this rabbit, VH1 is not expressed as it has been deleted. Analysis of cDNA clones from spleen of Alicia rabbits suggests that the expressed VHa-negative molecules also are encoded by a single germline VH gene. Thus, we suggest that nearly all rabbit VH regions are encoded by one to two germline VH genes and that antibody diversity is generated primarily by somatic hypermutation and gene conversion.

Amino Acid Sequence↗
PubMed1991

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit.

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the VH gene cluster up to, or 3' of, C mu. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entire VH cluster and upstream of the JH cluster within an approximately 50 kilobase (kb) region containing expanses of repetitive-sequence DNA as well as DH genes. DH-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5' of DH1 and 5' of DH2 genes; in the third it occurred 3' of the DH2 genes but at least approximately 5 kb 5' of the JH region.

Animals↗
PubMed1991

Patellar location changes following EMG biofeedback or progressive resistive exercises.

Changes in the patellofemoral congruence (PFC) angle, the patellar rotation (PR) angle, and the sulcus angle following EMG biofeedback training that emphasized vastus medialis obliquus strengthening, the Daily Adjustable Progressive Resistive Exercise (DAPRE) technique, or no exercise were studied. Subjects were 30 normal, college females. The EMG biofeedback group and the DAPRE group followed established protocols for a 3-wk training period. The control group refrained from exercise. Patellar angles were determined with x-rays both pre- and posttest with the quadriceps relaxed and contracted. Reliability of the x-rays was determined with a correlation matrix of pre- and posttest sulcus angle measurements (P less than 0.001). EMG biofeedback training resulted in significant changes in the PFC angle with the quadriceps contracted (P less than 0.05). DAPRE resulted in significant changes in the PR angle with the quadriceps contracted (P less than 0.05). The results of this study suggest that quadriceps group strength changes are not enough to fully rehabilitate patellar tracking dysfunctions. The use of EMG biofeedback training to selectively strengthen the vastus medialis obliquus appears to be essential in correcting faulty patellar tracking.

Adult↗
PubMed1990

Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits.

Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH1, raises the question of how rabbits generate antibody diversity. VDJ diversification was analyzed by cloning and sequencing VH1 gene rearrangements. Comparison of these sequences with that of germline VH1 identified clusters of nucleotide changes, including codon insertions and deletions. To investigate whether gene conversion was involved in this somatic diversification, we searched a data base of rabbit germline VH gene sequences for donor VH genes; potential donors were identified for five diversified regions. We conclude that somatic gene conversion has a major role in generating antibody diversity in rabbits. These studies provide clear evidence for somatic gene conversion of mammalian VDJ genes.

Animals↗
PubMed1990

Activation of the alternative pathway of complement by twelve different rabbit-mouse chimeric transfectoma IgA isotypes.

Previous experiments have resulted in the identification and cloning of 13 nonallelic genes encoding the constant region of rabbit IgA H chains. The genes, C alpha 1 to C alpha 13, were each cloned into an expression vector containing the VDJ gene of a dansyl (DNS)-binding murine hybridoma and the constructs were then transfected into SP2/0 cells that were producing murine kappa-L chains from the DNS-binding hybridoma. Of the 13 resulting transfectomas, 12 were shown, by ELISA, to secrete DNS-binding chimeric rabbit-mouse IgA molecules. These transfectoma antibodies, representing 12 different isotypes, are of high affinity and provide a unique source of Ag-specific IgA for comparison of the functions of the multiple IgA isotypes. One such function for antibodies is activation of C by either the classical or alternative pathway. We have used the DNS-binding IgA transfectoma antibodies in C assays based on binding of rabbit C3 to IgA-Ag complexes in an ELISA. The results demonstrated that all 12 IgA isotypes are capable of activating C by the alternative pathway but that none can activate C by the classical pathway. Control experiments demonstrated that activation was hapten dependent and was not caused by endotoxin contamination. These data demonstrate that Ag-specific IgA molecules, unmodified by heat or chemical aggregation, activate C by the alternative pathway but not by the classical pathway.

Animals↗
PubMed1990

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity.

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.

Alleles↗
PubMed1990

Restricted utilization of germ-line VH genes and diversity of D regions in rabbit splenic Ig mRNA.

Previous studies have suggested that the majority of rabbit germ-line VH genes encode molecules that are rarely found in serum or secretory Ig. To examine the repertoire of expressed VH genes, we prepared a cDNA library from splenic mRNA of an alpha 1/alpha 1 rabbit and isolated 10 complete VH-encoding cDNA clones. None of the cDNA clones hybridized to an oligomer that had hybridized to more than 50% of cloned germ-line VH genes. These data indicate that only a subset of germ-line VH genes are used in functional VDJ rearrangements. DNA sequence analysis demonstrated that the 10 cDNA clones contained highly similar VH regions, further suggesting that the repertoire of utilized VH genes is limited. In contrast, the D regions of each of the 10 clones exhibited little similarity to one another, suggesting that the rabbit has a large D region repertoire. We propose that the apparent lack of diversity within the VH segment of VDJ rearrangements is offset by extensive D region diversity.

Animals↗
PubMed1990

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination.

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.

Amino Acid Sequence↗
PubMed1990

Restricted utilization of VH and DH genes in leukemic rabbit B cells.

Polyclonal B cell leukemias have been generated in 17- to 20-day old Emu-myc transgenic rabbits. To analyze the repertoire of VH genes utilized in early B cells, eight VDJ genes were cloned from these leukemic cells. The nucleotide sequences of these genes indicated that seven of the eight VDJ genes encoded prototype VHa1, VHa2 or VHa3 allotypes. The two VDJ genes encoding VHa1 molecules had VH segments with identical nucleotide sequences; similarly, the VH segments of the four VDJ genes encoding VHa2 molecules were identical, with the exception of a single base pair. These data suggest that a limited repertoire of VH genes were utilized in the generation of these VDJ genes. The DH segments of these genes were limited to two DH families, D1 and D2, indicating that a restricted repertoire of DH genes also had been utilized. Since these leukemic cells probably developed early in ontogeny, we suggest that this restricted utilization of VH and DH genes is representative of B cells from developmentally immature rabbits.

Amino Acid Sequence↗
PubMed1990

Limited number of immunoglobulin VH regions expressed in the mutant rabbit "Alicia".

A unique feature of rabbit Ig is the presence of VH region allotypic specificities. In normal rabbits, more than 80% of circulating immunoglobulin molecules bear the VHa allotypic specificities, al, a2 or a3; the remaining 10% to 20% of immunoglobulin molecules lack VHa allotypic specificities and are designated VHa-. A mutant rabbit designated Alicia, in contrast, has predominantly serum immunoglobulin molecules that lack the VHa allotypic specificities (Kelus and Weiss, Proc. Natl. Acad. Sci. USA 1986. 83: 4883). To study the nature and molecular complexity of VHa- molecules, we cloned and determined the nucleotide sequence of seven cDNA prepared from splenic RNA of an Alicia rabbit. Six of the clones appeared to encode VHa- molecules; the framework regions encoded by these clones were remarkably similar to each other, each having an unusual insertion of four amino acids at position 10. This insertion of four amino acids has been seen in only 2 of 54 sequenced rabbit VH genes. The similarity of the sequences of the six VHa- clones to each other and their dissimilarity to most other VH genes leads us to suggest that the VHa- molecules in Alicia rabbits are derived predominantly from one or a small number of very similar VH genes. Such preferential utilization of a small number of VH genes may explain the allelic inheritance of VH allotypes.

Amino Acid Sequence↗
PubMed1989

The IgA heavy-chain gene family in rabbit: cloning and sequence analysis of 13 C alpha genes.

Southern analysis has previously shown that the rabbit genome contains multiple genes coding for the constant regions of IgA heavy chains. In the present study, clones containing these C alpha genes have been isolated from cosmid and phage libraries. Restriction mapping and Southern analysis of the clones identified 13 non-allelic C alpha genes; 11 of the genes were clustered in individual or overlapping clones. The clustered genes are separated by 8-18 kb, and in total, the C alpha genes span a minimum of 160 kb of DNA. Southern analysis has shown that all genes within a cluster have the same transcriptional orientation, and that switch sequences are present 5' of at least 12 of the 13 genes. The nucleotide sequence of each C alpha gene was determined, and it appears that all genes are functional; thus, rabbit may have as many as 13 IgA isotypes. Comparisons of the protein sequences encoded by the 13 C alpha genes showed that the CH2 and CH3 domains of the alpha-chains are highly conserved, whereas the CH1 and hinge regions are highly diverse. Southern analysis of genomic DNA samples from other species within the order Lagomorpha showed that all samples had multiple C alpha hybridizing fragments. Thus, it is likely that all lagomorphs have multiple IgA isotypes and hence complex secretory immune systems.

Amino Acid Sequence↗
PubMed1989

Identification of functionally important residues in the DNA binding region of the mnt repressor.

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.

Amino Acid Sequence↗
PubMed1989

The Arc and Mnt repressors. A new class of sequence-specific DNA-binding protein.

Genetic, biochemical, and biophysical studies have begun to reveal details of the structures of Arc and Mnt and show that these repressors use residues at their N-terminal ends for operator recognition and binding. Some of the DNA contacts made by these residues have been identified, and this information together with NMR studies has permitted the construction of models of the DNA binding region. Although the accuracy of these models remains to be determined, it seems clear that Arc and Mnt are members of a new class of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1989

Ig VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA.

Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.

Amino Acid Sequence↗