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A survey of diagnostic details is made on which functional analysis is based. Symptoms connected with the temporo-mandibular joint, muscle palpation, radiological demonstration of the temporo-mandibular joint, and registration of the positional jaw relation are especially dealt with. The methods of examination which are prerequisite to a safe diagnosis are critically evaluated.
Functional analysis of human papillomavirus type 16 E7 protein by complementation with adenovirus E1A mutants in baby rat kidney cells has shown that the retinoblastoma gene product (RB)-binding region of E7 can substitute in trans for that of E1A. An N-terminal E7 mutant was unable to complement an E1A mutant unable to bind p300, indicating that the two mutants were defective for functionally equivalent activities. E7 proteins with mutations within the RB-binding region were also unable to complement either the non-p300-binding E1A mutant or the N-terminal E7 mutant, suggesting that these mutations affect more than just RB binding.
Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations.
We have demonstrated previously that transfer function analysis can be used to precisely characterize the respiratory sinus arrhythmia (RSA) in normal humans. To further investigate the role of the autonomic nervous system in RSA and to understand the complex links between respiratory activity and arterial pressure, we determined the transfer functions between respiration, heart rate (HR), and phasic, systolic, diastolic, and pulse arterial pressures in 14 healthy subjects during 6-min periods in which the respiratory rate was controlled in a predetermined but erratic fashion. Pharmacological autonomic blockade with atropine, propranolol, and both, in combination with changes in posture, was used to characterize the sympathetic and vagal contributions to these relationships, as well as to dissect the direct mechanical links between respiration and arterial pressure from the effects of the RSA on arterial pressure. We found that 1) the pure sympathetic (standing + atropine) HR response is characterized by markedly reduced magnitude at frequencies greater than 0.1 Hz and a phase delay, whereas pure vagal (supine + propranolol) modulation of HR is characterized by higher magnitude at all frequencies and no phase delay; 2) both the mechanical links between respiration and arterial pressure and the RSA contribute significantly to the effects of respiration on arterial pressure; 3) the RSA contribution to arterial pressure fluctuations is significant for vagal but not for sympathetic modulation of HR; 4) the mechanical effects of respiration on arterial pressure are related to the negative rate of change of instantaneous lung volume; 5) the mechanical effects have a higher magnitude during systole than during diastole; and 6) the mechanical effects are larger in teh standing than the supine position. Most of these findings can be explained by a simple model of circulatory control based on previously published experimental transfer functions from our laboratory.
We conducted a brief functional analysis to identify maintaining variable for aggressive behavior and an alternative replacement response during a 90-min outpatient evaluation of 3 individuals with severe handicaps. During the initial analogue assessment, which focused on identifying maintaining contingencies for aggressive behavior, each participant displayed a substantially greater frequency of aggressive behavior during one condition than during any other. The contingency that produced the highest percentage of aggressive behavior was then presented for the occurrence of a specific alternative behavior (a mand). During this contingency reversal phase, each participant displayed a substantial reduction in aggressive behavior and a substantial increase in alternative behavior, thus providing a direct analysis of the equivalency of the contingency for maintaining either behavior.
A functional analysis of the Aspergillus nidulans 3-phosphoglycerate kinase pgk promoter was undertaken using gene fusions to the lacZ gene of Escherichia coli, and introducing these into a beta-galactosidase-deficient strain of A. nidulans. Expression of a particular gene fusion in transformed strains depends upon the site of integration of the vector into the genome, and when specifically targeted to the catabolic quinate dehydrogenase qutE (selective marker) locus is directly proportional to its copy number. The analysis of transformed strains with single copies of pgk promoter deletion--lacZ fusions at the qutE locus identified three constitutive, positively acting sequence elements in the pgk gene. Sequence located between -161 and -120 nucleotides relative to the transcript start site +1, and including an element with a seven-out-of-eight nucleotide match (AAGCAAAT; -131 to -124) to the consensus eukaryotic octamer sequence ATGCAAAT, is essential for expression, and deletion of the complete 41-nucleotide sequence abolishes transcription. Sequence encompassing codons 14 to 183 and including the two introns of pgk contributes approximately one-third of the total activity, and far upstream sequence 5' to position -638 contributes approximately a further one-third total activity. In addition, sequence located -638 to -488 nucleotides, which includes an apparent consensus feature of A. nidulans glycolytic genes, affects carbon source-dependent regulation of expression. This region is required for an approximately 50% increase in pgk expression when A. nidulans is grown on gluconeogenic compared with glycolytic carbon sources.
This study was undertaken to determine whether it was possible to use a multivariate discriminant function analysis to assess the severity of coronary artery disease in patients with angina pectoris. One hundred and forty-three patients diagnosed as having angina pectoris who formed a 'learning group' were subjected to an exercise test on a treadmill using a Bruce protocol. The items of data from the test least likely to be influenced by beta blockade were considered for the analysis along with two discrete variables (the gender and the use of beta blockers). All the patients subsequently underwent coronary angiography and the severity of the coronary artery disease was graded according to a scoring system. In addition, patients in 'learning group' were classified into two 'Groups', (I) those with normal vessels or with disease in one or two of the major arteries, (II) those with disease in the left main stem or in all three major arteries. The multivariate analysis yielded a score based upon 4 factors: (i) gender, (ii) percentage increase in double product at the highest level of exercise achieved, (iii) duration of ST depression in the recovery period and (iv) the summation of the ST depression in the standard electrocardiogram excluding lead aVR. There was a significant overall correlation between the discriminant function score and the coronary artery score for severity of the disease. In addition, it was found that, of patients having discriminant function score below 0, 96% (69/72) were in Group I. The post-test risk of a positive test was 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)
Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.
Analysis of the functional nature of mutations can be based on their manifestation in organisms with a deletion or a duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H it is feasible to produce mice with tertiary trisomy for proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t-complex tail interaction) and tcl (t complex lethal), that are specific for t haplotypes. The set of dominant and recessive mutations was assigned to two groups, one obligatory manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself, depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes which is to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.
Analysis of the functional nature of mutations can be based on comparisons of their manifestation in organisms with a deletion or duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H, it is feasible to produce mice with tertiary trisomy of the proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t complex tail interaction), and tcl (t complex lethal) that are specific to t haplotypes. The set of dominant and recessive mutations was assigned to two groups: one obligatory, manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes. It seeks to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.
Phenotypic and functional analysis was performed with lamina propria mononuclear cells (LPMCs) isolated from colonoscopic biopsies in 27 patients with ulcerative colitis (UC). The proportion of T lymphocytes displaying HLA-DR antigens, interleukin 2 (IL-2) receptor, and transferrin receptor was greater in active UC than in control diseases. When LPMCs were cultured with IL-2 or phytohemagglutinin for 72 h, there were no significant differences in the proportion of cells bearing these activation markers between active UC and controls. The proportion of CD56+ cells and lymphokine-activated killer (LAK) cell activity was lower in LPMCs from active UC than in control cells, and depletion of CD56+ cells from control lamina propria cells essentially eliminated LAK cell activity. Mucosal T lymphocytes may be activated in vivo during active inflammation in UC, and lower levels of intestinal LAK cell activity may be related to the decrease of CD56+ cells under these conditions.
A phenotypic and functional analysis has been made of the cellular response in regional lymphoid tissue of C57BL/6J mice infected with lymphocytic choriomeningitis virus. Massive recruitment of nondividing cells occurred from 3 days after infection, with total numbers of CD8+ T lymphocytes, B220+ B cells, and Thy-1- B220- null cells being high from day 4 to day 6. In contrast, the peak counts for CD4+ T cells were recorded on day 4 and declined dramatically thereafter. Enhanced expression of IL-2R and Ly-24, both of which can be regarded as T cell activation markers, was found for both the CD4+ and the CD8+ subsets, being most prominent for the CD8+ T cells on day 6. Evidence of T cell proliferation was not recognized until days 5 and 6, coincident with enhanced responsiveness of the lymphocytes to rIL-2 and the development of virus-specific cytotoxic activity. Elimination of the CD4+ T cells by treatment of mice with mAb did not modify either the pathogenesis of lymphocytic choriomeningitis, or the expression of activation markers on the CD8+ T cells which are known to be the key effectors in this disease. Thus, the pattern of responsiveness for the CD8+ population is of recruitment to the lymph node, progressive increase in the expression of activation markers and enhanced sensitivity to rIL-2, with late proliferation and generation of cytotoxic activity. This model provides a system for the rigorous in vivo analysis of parameters influencing lymphocyte differentiation and activation in a virus infection.
Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.
Several novel, structurally distinct classes of specific human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) nonnucleoside inhibitors have been described recently. These include the pyridinone derivatives L-697,639, L-697,661, and L-696,229 as well as BI-RG-587 and the tetrahydroimidazo[4,5,1-j,k]-benzodiazepin-2(1H)-one and -thione compounds. Previous studies have implicated involvement of the RT amino acid residues at positions 103, 181, and 188 in the activity of the compounds. Accordingly, HIV-1 RT mutants containing a series of amino acid substitutions at these positions were constructed. The relative resistance of purified mutant enzymes to each of the inhibitors was assessed. This analysis established the functional equivalence of the three inhibitor classes and provided evidence for the interaction of the 103 site with the 181/188 region. Amino acid substitutions at these positions were also found to influence RT sensitivity to inhibition by phosphonoformate, thereby suggesting a close association between this pyrophosphate analog's binding site in RT and the binding site of the nonnucleoside inhibitors. In addition, aromatic stacking of the amino acid side groups at residues 181 and 188 was suggested to be required for inhibitor activity.
The present study describes the phenotypic and functional analysis of tumor-infiltrating lymphocytes isolated from a germinoma located in the pineal region. Tumor-infiltrating lymphocytes were separated from the germinoma and cultured in medium containing IL-2 (1000 U/ml). An immunohistochemical analysis of frozen sections revealed that 90% of the germinoma-infiltrating lymphocytes were CD3-positive T cells expressing CD4, CD8, and HLA Class I and Class II antigens, but were negative for CD16, CD20, CD23, CD25 and CD14 antigens. After in vitro cultivation in the presence of high concentrations of IL-2, the lymphocytes proliferated for 2 weeks, showing marked DNA synthesis. In addition, the lymphocytes could lyse NK-resistant allogeneic target cells. These results provide evidence for a potential role of germinoma-infiltrating lymphocytes in vivo, and suggest that the lymphocytes may control the growth of autochthonous tumor cells by killing those that are not restricted to the major histocompatibility complex.
We undertook a structure--function analysis of human tissue plasminogen activator (tPA) using linker-scanning and deletion mutagenesis. Synthetic oligonucleotide linkers were introduced into the tPA cDNA at pre-existing restriction enzyme sites. This generated a series of tPA variants which contained small primary sequence alterations consisting of point mutations, deletions or insertions. The majority of the linker-insertion variants demonstrate a significant reduction in amidolytic and fibrinolytic activity in comparison to wild-type tPA. The exceptions are the variants with linker-inserts placed at the BglII(115) and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively), which encode insertions at the boundaries of the finger domain. The variants with linker-inserts in the light chain (protease domain) of tPA are the lowest in enzymatic activity. Particularly sensitive to mutation are highly conserved amino acids. Heavy chain deletion variants were constructed from point mutants at the domain boundaries of tPA. Deletion of the kringle domains lowers the fibrinolytic activity to a greater extent than deletion of the finger or growth factor domains. We conclude that alterations in any domain of the tPA molecule, and particularly in the highly conserved residues within these domains, can affect fibrinolytic activity.
We present here a functional analysis of ARGRI and ARGRIII regulatory proteins which are involved together with ARGRII in specific regulation of arginine anabolic and catabolic pathways. Unlike ARGRII, ARGRI and ARGRIII have no transcriptional activation capacity. The first 60 amino acids of ARGRI (out of 177) are dispensable for its activity. The functional domain of the protein is located in the region of homology with MCM1 and SRF proteins. ARGRIII contains in its C-terminal portion a stretch of 17 aspartate residues which are indispensable for arginine regulation. Gene disruption of the ARGRIII gene impairs the growth of the mutant on rich medium, showing that ARGRIII has a pleiotropic role in the cell.
The dual MAS1'-2' promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2' end relative to the corresponding 1'. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1' promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1'-2' TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.