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Biomedical subjects

K L Knight

Publications and source records attributed to K L Knight.

At least 55 records · Page 3Linked to original sources
PubMed1996

Evidence for limited B-lymphopoiesis in adult rabbits.

Rabbits are born with a limited VDJ gene repertoire formed primarily by rearrangement of one VH gene, VH1. The VDJ genes are undiversified at birth but become diversified by approximately 2 mo of age. To investigate more closely the time during which this diversity occurs, we determined the nucleotide sequences of VDJ genes from peripheral blood leukocytes taken from young rabbits at various time points, and we examined the extent of the diversification of the VDJ genes. At 4 wk of age there were, on average, 3 nucleotide changes per VH region, with approximately 75% of the genes showing some diversification. The number of nucleotide changes per VH region increased to 12 by 6-8 wk of age, and all but 1 of the 35 sequences analyzed were diversified. Because only a limited number of genes can be examined by nucleotide sequence analysis, we used an RNase protection assay to examine a large number of genes and we determined the level of undiversified VH1 mRNA in lymphoid organs of both young and adult rabbits. In young rabbits, we found a high level of undiversified VDJ genes, but the level was greatly reduced by 2 mo of age. By adulthood, essentially all VDJ genes of cells from appendix, peripheral blood, and bone marrow were diversified. Because we had expected B lymphopoiesis to be ongoing in the bone marrow of adult rabbits, we were surprised not to find undiversified VDJ genes from the newly generated B cells. Therefore, we searched for evidence of ongoing B lymphopoiesis in bone marrow by isolating and examining circular DNA for the presence of VD and DJ recombination signal joints. We found highly reduced levels of recombination signal joints in bone marrow of adult rabbits relative to the levels found in bone marrow of newborn rabbits. These data indicate that limited VD and DJ gene rearrangements occur in bone marrow of adult rabbits, and we therefore suggest that B lymphopoiesis is limited in adults.

Aging↗
PubMed1996

Cooling does not affect knee proprioception.

The effect of cooling on proprioception of the knee has not been studied extensively. In this study, we investigated the movement reproduction (timing and accuracy) aspect of proprioception. Subjects were tested under two conditions: a 20-minute application of ice and control. Proprioceptive accuracy and timing were measured by passively moving the knee, then comparing the subject's active reproduction of the passive movement. Subjects were blindfolded, then tested in three sectors of the knee's range of motion: 90 degrees to 60 degrees , 60 degrees to 30 degrees , and 30 degrees to full extension. Ice application had no apparent effect on the subject's ability to perform accurate movement reproductions in the sectors tested. However, accuracy of the subject's final angle reproduction varied between the sectors as did the total time of the movement. One possible explanation for the difference between sectors is that different receptors are active at different points in the knee's range of motion. We conclude that cooling the knee joint for 20 minutes does not have an adverse effect on proprioception.

Journal Article↗
PubMed1996

Structure of a scholarly manuscript: 66 tips for what goes where.

OBJECTIVE: TO SHARE WITH POTENTIAL AUTHORS TIPS FOR CONSTRUCTING A SCHOLARLY MANUSCRIPT AND FOR ORGANIZING INFORMATION IN VARIOUS TYPES OF SCHOLARLY MANUSCRIPTS: experimental reports, literature reviews, case reports, and clinical techniques. DESCRIPTION: The goal of writing a scientific/technical/ medical article is to communicate new information that hopefully has clinical relevance and will improve health care. This information must be organized and presented clearly and logically. We present 66 tips for organizing a scholarly manuscript. We tell not only what goes where in the manuscript but also how to construct each of the elements so as to logically communicate the author's message. The tips are numbered to facilitate referencing. CONCLUSION: By becoming familiar with these tips, potential authors can avoid making mistakes that may hinder publication of their manuscripts.

Journal Article↗
PubMed1996

Optimizing scholarly communication: 30 tips for writing clearly.

OBJECTIVE: To share with potential authors tips for communicating their ideas more clearly in a scholarly manuscript. DESCRIPTION: Communicating scientific, technical, or medical information so that readers can understand its meaning requires logical organization and proper use of language. These 30 tips review basic English grammar and suggest ways authors can clearly and concisely present their material. We admonish authors to avoid common errors such as writing in the passive voice, overusing abbreviations, and emphasizing unimportant facts. CONCLUSION: Attention to matters of writing style enhances clear communication, which must be the prime objective of scientific writing.

Journal Article↗
PubMed1996

Ankle and thigh skin surface temperature changes with repeated ice pack application.

OBJECTIVE: Most of the research on cold applications has been performed on nonexercising supine subjects during a single cold pack application. Most athletic injuries occur during exercise, which increases skin temperature. Exercise before ice application will also increase ankle skin temperature during the rewarming phase. The purpose of this study was to examine the effects of activity on subsequent ice pack applications and rewarming using standard immediate care procedures. DESIGN AND SETTING: Three experimental conditions (20-, 30-, and 40-minute ice pack applications) were applied to 12 subjects in a repeated measures design. Subjects rode a bicycle ergometer for 15 minutes before ice application to the ankle and opposite thigh, and were active (walking with crutches, simulated showering and dressing) for 20 minutes following application. Subjects rested with the limb elevated for an additional 40 minutes. Ice packs were then reapplied for the appropriate time (20, 30, or 40 minutes) followed by 60 minutes of rest with the limb elevated. SUBJECTS: Twelve (8 males, 4 females) college-aged volunteers. Only subjects with good-to-high fitness levels were accepted for this study. MEASUREMENTS: Ankle skin, thigh skin, and atmospheric temperatures were measured every minute using an Isothermex (Columbus Instruments, Columbus, OH). RESULTS: Thigh temperature changes during the first ice application were greater during the 30- and 40-minute conditions than the 20-minute condition. Ankle and thigh temperature changes during the first ice application and rewarming, and for the entire trial were greater during the 40-minute condition than the 20-or 30-minute conditions. Throughout the first ice application and rewarming, and the entire trial, thigh temperature changes were greater during the 30-minute condition than the 20-minute condition. CONCLUSIONS: During immediate care procedures following injury, ice packs should be reapplied immediately following showering, changing clothes, and returning home.

Journal Article↗
PubMed1995

Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.

Animals↗
PubMed1995

Transchromosomally derived Ig heavy chains.

During an immune response, activated B cells undergo isotype switching and begin to express isotypes other than IgM and IgD. Isotype switching occurs when downstream C gamma, C alpha, or C epsilon genes are rearranged into the S mu chromosomal region, resulting in the deletion of the region in between. These rearrangements usually occur in cis, i.e., intrachromosomally. In previous studies, we analyzed allotypic specificities of rabbit secretory IgA and identified a substantial number of IgA heavy chains with VH and C alpha allotypes that were encoded by VH and C alpha genes in trans. In those studies, however, we could not determine whether the trans association of VH and C alpha occurred during VDJ gene rearrangement or during isotype switching. Here, we cloned rabbit cDNA which encodes these trans IgA heavy chains and determined the chromosomal origin of the VH, JH, and C alpha regions. To determine whether the trans association occurred during VDJ gene rearrangement, we analyzed the nucleotide polymorphism of the JH region and the VH allotype encoded by the cDNA. We found that the VH and JH genes used in the VDJ gene rearrangements were from the same chromosome, indicating that the VH, D, and JH gene rearrangements occurred in cis. Furthermore, we analyzed the DNA polymorphisms of JH and C alpha and showed that the VDJ and C alpha genes encoding the trans IgA molecules were derived from different parental chromosomes. We suggest that the trans association occurred during isotype switching. This study shows that VH and CH can associate transchromosomally as part of a normal immune response.

Amino Acid Sequence↗
PubMed1995

Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities.

The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro67-->Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of lambda cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)6 and (dT)6, as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro67-->Lys) and homologous recombination (Pro67-->Lys and Pro67-->Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Pro67 is located in a functionally important position in the RecA structure.

Amino Acid Sequence↗
PubMed1995

Fetal VDJ gene repertoire in rabbit: evidence for preferential rearrangement of VH1.

B cells in rabbit preferentially utilize the 3'-most VH gene, VH1, in VDJ genes. To determine whether the preferential utilization of VH1 results from preferential rearrangement, we examined VH gene usage in nonproductive VDJ gene rearrangements, genes that would not be influenced by antigen selection. Since nonproductive VDJ gene rearrangements are found infrequently in neonatal and adult rabbits, we examined the VDJ gene rearrangements during early fetal development. According to nested-primer polymerase chain reaction analyses, VDJ genes in fetal liver appeared around day 14 of gestation and nucleotide sequence analyses of VDJ genes cloned from 14- to 28-day-old fetuses showed that many of the genes were nonproductive. We found that most of these nonproductive genes had utilized VH1 indicating to us that the preferential utilization of VH1 is due to preferential rearrangement. We compared the nucleotide sequence of the promoter region of VH1 with sequences of several other unutilized and infrequently utilized VH genes and found that the core transcriptional factor motifs were similar in all VH promoters examined. We suggest that transcriptional activity of the VH1 promoter region does not explain the preferential rearrangement of VH1.

Animals↗
PubMed1995

Dramatic changes in DNA-binding specificity caused by single residue substitutions in an Arc/Mnt hybrid repressor.

Arc and Mnt are homologous repressors which recognize operator sequences that differ at 8-10 important positions. Nevertheless, single residue changes in an Arc/Mnt hybrid protein can switch DNA-binding specificity between the two operators and even allow one particular hybrid to bind strongly to both operators. The ability of single residue changes to radically alter binding specificity involves: 'master' residues that mediate some base contacts directly and some base contacts indirectly through residue-residue hydrogen bonds; identical residues which can make alternative sets of DNA contacts in the two operators; and amplification of the effect of each mutation because the proteins bind operator DNA as tetramers.

Amino Acid Sequence↗
PubMed1995

Agility following the application of cold therapy.

Cold application is commonly used before strenuous exercise due to its hypalgesic effects. Some have questioned this procedure because of reports that cold may reduce isokinetic torque. However, there have been no investigations of actual physical performance following cold application. The purpose of this study was to determine if a 20-minute ice immersion treatment to the foot and ankle affected the performance of three agility tests: the carioca maneuver, the cocontraction test, and the shuttle run. Twenty-four male athletic subjects were tested during two different treatment sessions following an orientation session. Subjects were tested following a 20-minute 1 degrees C ice immersion treatment to the dominant foot and ankle and 20 minutes of rest. Following each treatment, subjects performed three trials of each agility test, with 30 seconds rest between each trial, and 1 minute between each different agility test. The order in which each subject performed the agility tests was determined by a balanced Latin square. A MANOVA with repeated measures was used to determine if there was an overall significant difference in the agility times recorded between the cold and control treatments and if the order of the treatment sessions affected the scores. Although the mean agility time scores were slightly slower following the cold treatment, cooling the foot and ankle caused no difference in agility times. Also, there was no difference resulting from the treatment orders. We felt that the slightly slower scores may have been a result of tissue stiffness and/or subject's apprehension immediately following the cold treatment. Cold application to the foot and ankle can be used before strenuous exercise without altering agility.

Journal Article↗
PubMed1995

Sensory information can decrease cold-induced pain perception.

Specific terms are often used to describe the pain athletes typically experience during cold treatments. It is not clear whether providing athletes with such descriptive sensory information will decrease their perceived pain during treatments. The purpose of this study was to determine if subjects' perceptions of cold-induced pain could be influenced by the type of information provided before treatment, such as "pain will be cursing" or "pain will be flickering." Ninety Division I intercollegiate athletes were randomly assigned to one of five groups: traditional terms (cold, burning, aching, numbness), high-level terms (freezing, crushing, pounding, heavy), moderate-level terms (cold, gnawing, pulsing, aching), or low-level terms (cool, pinching, flickering, dull) from the McGill Pain Questionnaire, or no terms at all (control). The four groups that received a set of terms were told that those terms described the sensations they would feel during cold immersion of the ankle. Pain was measured with the McGill Pain Questionnaire every 3 minutes during a 21-minute immersion (1 degrees C) of the foot and ankle. Sensory, affective, evaluative, and miscellaneous pain measures were derived. The control group experienced greater sensory and affective pain than did any of the other groups and experienced greater evaluative pain than did the groups receiving low-level or traditional terms. We conclude that providing athletes with some type of sensory information to describe their cold-pain experience will decrease their perceived pain during cold immersion, although it does not seem to matter what terms are used.

Journal Article↗
PubMed1995

A comparison of isokinetic and isotonic predictions of a functional task.

Controversy still exists regarding the use of isokinetic and isotonic exercise in rehabilitation. Many authors have compared these two types of training methods on various strength measures and functional activity, but have used open kinetic chain training. The purposes of this investigation were to determine: 1) which form of closed kinetic chain training, isokinetic or isotonic, would produce the greatest increase in one-legged jump reaction force, and 2) which training method most accurately predicts peak force produced during a one-legged jump. Forty-two legs from 21 female volunteer subjects were used. Each subject had her dominant and nondominant extremities identified, and then each extremity was randomly assigned to either isokinetic training, isotonic training, or control. Both training groups trained using a leg press exercise 3 days a week for 5 weeks, while the control extremities did not train. The isokinetic extremities were trained using a velocity spectrum (two sets of 10 repetitions at each speed: 60 degrees , 180 degrees and 240 degrees /sec) and the isotonic extremities trained using the DAPRE technique. Data were analyzed with an analysis of variance (ANOVA). There was no difference between the three groups for change in one-legged jump force. Both isokinetic and isotonic groups increased strength after training, but these changes did not correlate with changes in one-legged jump reaction force. These results suggest that changes in neither isokinetic force nor isotonic weight lifted developed in a nonweight-bearing closed kinetic chain, directly translate into increased force production during a functional activity.

Journal Article↗
PubMed1994

Solution structure of dimeric Mnt repressor (1-76).

Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.

Amino Acid Sequence↗
PubMed1994

Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities.

Using a combinatorial cassette mutagenesis procedure we have introduced a large number of single and multiple amino acid substitutions into an area of the RecA protein defined by residues 152-159. This sequence overlaps the disordered loop 1 region (L1) in the RecA crystal structure which has been hypothesized to be involved in DNA binding. Assays for recombinational DNA repair and LexA coprotease activities identify Glu154 as the only one of these 8 residues which is critical to RecA function. Several other mutations observed at nearby residues support the identity of Glu154 as the most important of the 14 residues in the area defined by Pro151 to Met164. In addition, Gly157 and Glu158 appear to be hot spots for the occurrence of mutation-induced constitutive coprotease activity.

Amino Acid Sequence↗
PubMed1994

Preferential VH gene usage in rabbit Ig-secreting heterohybridomas.

B cells from leukemic rabbits preferentially use a single VH gene, VH1, in their VDJ gene rearrangements. To determine whether Ig-secreting B cells from normal rabbits also preferentially use VH1, we generated rabbit X mouse heterohybridomas that stably secreted rabbit Ig that expressed VHa allotypic specificities and analyzed the VH genes used in their VDJ gene rearrangements. We cloned the VDJ genes from nine heterohybridomas, and by comparing the restriction map of the DNA immediately 5' of the translational start site of these VDJ genes to that of the same region 5' of VH1, we showed that eight of the nine heterohybridoma clones use VH1. Comparison of the nucleotide sequences of the eight VH1-using VDJ genes with the nucleotide sequence of germ-line VH1 showed that each of them had somatically diversified. The diversified regions included clustered nucleotide changes and codon insertions and deletions, such as would be expected if the diversification process involved somatic gene conversion. We searched our database of germ-line VH genes for genes that could serve as donors for the gene conversion events, and we identified potential VH donor genes for five regions of diversification. One of these regions of diversification spanned at least 132 bp and included a codon insertion as well as 15 nucleotide changes. The data confirm that Ab diversity may be generated by a somatic gene conversion-like mechanism. The results directly demonstrate that Ig-secreting B cells from normal rabbits preferentially use VH1 in their VDJ gene rearrangements.

Amino Acid Sequence↗
PubMed1994

Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site.

We have introduced a large number of single amino acid substitutions at six positions that lie within the P-loop motif of the ATP binding site in the Escherichia coli RecA protein. The activity of each recA mutant was determined using genetic assays which assess the catalytic proficiency of the RecA protein for both homologous genetic recombination and recombinational repair of damaged DNA. The six residues displayed unique patterns of allowed versus non-allowed substitutions that define the functional and structural constraints at each position. Our results show that while the restricted mutability of Gly66 and Ser70 conform to expectations based on their positions in the RecA crystal structure, strict constraints are in effect at positions 68 and 69 that are not apparent in the RecA structure but would be compatible with the proximity of these side-chains to bound DNA. Thr74 shows a rather unexpected pattern of allowed substitutions that may reflect two distinct structural solutions to optimal stabilization of bound nucleotide. In addition, specific substitutions at Pro67 result in mutant RecA proteins that appear to discriminate between homologous genetic recombination and recombinational DNA repair.

Adenosine Triphosphate↗