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PubMed1990

DNA-sequence and metal-ion specificity of the formation of *H-DNA.

The homopyrimidine-homopurine sequence d(CT/GA)22 undergoes, in the presence of zinc ions, transition to an altered DNA conformation (*H-DNA) which is neither H-DNA nor B-DNA. *H-DNA is characterized by a peculiar chemical reactivity pattern in which most of the polypyrimidine strand is hyperreactive to osmium tetroxide and the central part of the polypurine strand is sensitive to diethylpyrocarbonate. Formation of *H-DNA is specific of metal-ion. *H-DNA is detected in the presence of Zn++, Cd++ and Mn++. The efficiency on promoting the transition is in the order of Zn++ greater than Cd++ much greater than Mn++. Formation of *H-DNA is also specific of nucleotide sequence. From all the different homopolymeric sequences tested only the d(CT/GA)22 sequence showed the zinc-induced transition to *H-DNA. These results suggest that stabilization of *H-DNA involves the formation of a specific complex between the metal-ion and the nucleotide sequence. The biological relevance of these results is discussed in view of the important role that zinc ions play on many nucleic acids processes.

Base Sequence↗
PubMed1989

Syphilis.

The clinical manifestations of syphilis can resemble many diseases. The diagnosis can be confirmed through serologic tests and by direct microscopic observation of Treponema pallidum, subspecies pallidum in lesion, biopsy, or autopsy specimens. Direct microscopic examination may be carried out by darkfield methods or by immunofluorescent staining. The serologic testing sequence is usually a screening nontreponemal test followed by confirmation with a specific treponemal test. Serologic tests for syphilis have been highly standardized in the past, but today quality control lies in the hands of the laboratorian.

Humans↗
PubMed1988

Human immunodeficiency virus test evaluation, performance, and use. Proposals to make good tests better.

Human immunodeficiency virus (HIV) tests are essential for detecting asymptomatic infection and are helpful in confirming the diagnoses of acquired immunodeficiency syndrome-related complex and acquired immunodeficiency syndrome. Nonetheless, many aspects of their use remain controversial, partly because of concerns about test accuracy. This article reviews the scientific basis for the evaluation, performance, and use of the most commonly employed HIV assays. Current test performance could be improved by better standardization of test procedures and institution of mandatory proficiency testing and licensure of clinical laboratories that perform HIV testing. Test utility could be enhanced by sequencing tests more appropriately and by interpreting test results in conjunction with the clinical purpose for which the test is being used and the characteristics of the population under study. Finally, HIV tests should be evaluated in a manner that minimizes spectrum and referral bias and inadequate reference standard confirmation, problems that have affected the evaluation of current tests.

Acquired Immunodeficiency Syndrome↗
PubMed2026

Longitudinal genome-wide aneuploidy measurements in circulating cell-free DNA to predict lack of benefit from pembrolizumab in patients with metastatic urothelial cancer.

Accurate prediction of lack of benefit from pembrolizumab in patients with metastatic urothelial cancer (mUC) is an unmet need. We investigated the dynamics of circulating tumor DNA (ctDNA) load, estimated using the modified fast aneuploidy screening test-sequencing system (mFast-SeqS), as a potential biomarker for early on-treatment identification of treatment response. A total of 104 patients with mUC treated with pembrolizumab from two prospective biomarker discovery trials were included and mFast-SeqS was performed on paired blood samples collected at baseline and on-treatment. Patients with a high on-treatment aneuploidy score (&#x2265;&#x2009;5, n&#x2009;=&#x2009;26) had a shorter median OS than patients with a low (<&#x2009;5) score (n&#x2009;=&#x2009;76) (3 vs 17&#x2009;months: P-value<&#x2009;0.001). Patients with an increased (n&#x2009;=&#x2009;10), stable (n&#x2009;=&#x2009;66), or decreased (n&#x2009;=&#x2009;28) on-treatment score relative to their baseline score had a median PFS of 1.5, 4.0, and 8.3&#x2009;months, respectively. Median OS was 3.0, 11.1, and 18.7&#x2009;months, respectively. In patients with mUC treated with pembrolizumab, the on-treatment mFast-SeqS-based ctDNA level and its dynamics relative to baseline are independent prognostic markers that can be used to identify patients that are unlikely to benefit from pembrolizumab.

Humans↗
PubMed1975

Calcium-binding protein of bovine intestine. The complete amino acid sequence.

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.

Amino Acid Sequence↗
PubMed1988

Functional analysis of mitochondrial protein import in yeast.

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of the mitochondrial transcription factor mtf2 of Saccharomyces cerevisiae.

The nuclear gene for a new mitochondrial transcription factor (mtf2) was isolated by transformation of mutant pet-ts3504. It was localized on a 6.4 kb fragment of yeast genomic DNA by subcloning and complementation tests. Sequencing of a 1.7 kb DNA fragment revealed an open reading frame of 1320 bp. A transcript of 1400 nucleotides can be assigned to this region. Gene disruption of this reading frame in a wild-type yeast strain created a stable pet phenotype. Further analysis of this insertion mutation showed that it is allelic to the mutated gene of pet-ts3504. Comparison of the 5' upstream regions of MTF2 and a previously characterized mitochondrial transcription factor (MTF1) revealed common sequence motifs which may be important for coordinated regulation of gene expression.

Alleles↗
PubMed1990

Pharmacological testing of growth hormone secretion.

The laboratory confirmation of growth hormone (GH) deficiency (GHD) has been extensively studied. Multiple stimuli induce GH release, but insulin-induced hypoglycemia usually is considered the 'gold standard'. Seventy-five to 90% of normal children have significant increments of hGH to any single test. Complete and partial syndromes of GHD have been defined, but some patients with a clinical appearance of GHD release hGH during provocative testing. Discordant results on varied tests may occur in the same child. Sequential and simultaneous tests have been attempted with diverse time patterns; testing sequence may significantly affect data interpretation. Persistent problems with GH provocative tests remain: normal data not strictly defined throughout childhood, multiple tests with discordant results, and substantial discrepancies of immunopotency estimates with different radioimmunoassays. Some children with 'normal' hGH increments during provocative tests, despite clinical GHD, may require short-term treatment with hGH to finally establish the diagnosis.

Arginine↗
PubMedDate not supplied

Assessing the limits of knee motion.

Ten patients were examined clinically by 11 experienced knee surgeons and with three measurement devices (Genucom, Knee Signature System, and KT-1000). Nine of the 10 patients examined had sustained documented intraarticular ligament disruptions including eight ACL disruptions and five posterior cruciate ligament disruptions. The eight patients with ACL injuries had undergone reconstructive surgery. The clinical examination test sequence was performed as described by the International Knee Documentation Committee. Testing results were reported as estimated degrees of angulation or millimeters of displacement. Testing results were divided into two groups based on the index minus nonindex knee difference. Eighteen displacement tests were performed on each knee by each examiner. The index minus nonindex knee difference was greater than 3 mm in 16% of the tests. Only 1 of the 198 displacement tests performed on the subject who did not have a ligament disruption was recorded as an index minus nonindex difference greater than 3 mm. There was appreciable difference between measurements recorded by different examiners, particularly anterior/posterior measurements in patients who had sustained combined anterior and posterior cruciate ligament injuries. There is a need to rigorously evaluate limits of motion tests that are used to develop treatment plans and report the results of ligament surgery.

Adult↗
PubMed1976

Computerized, noninvasive tests of lung function. A flexible approach using mass spectrometry.

The design, operation, and some applications of a computerized pulmonary function testing system built around a mass spectrometer are described. The test sequence, performed in 10 to 20 min, includes spirometry, a single-breath N2 washout, and measurement of the diffusing capacity of the lung for CO. Secondary tests, an integral part of the sequence, include rebreathing estimates of lung volume and cardiac output, and a breath-by-breath analysis of over-all gas exchange. These secondary tests lead to computer modeling of a one-compartment lung closely matched to the subject's lungs. Differences between alveolar plateau slopes in the model and real lung provide information about the degree of ventilation-perfusion mismatch in the subject. It is expected that the combination of tests will be useful in the early detection of lung disease.

Computers↗
PubMed1977

The detection of sympatric sibling species using genetic correlation analysis. I. Two loci, two gamodemes.

Four models are presented describing zygotic frequencies at two loci for one or two sympatric but genetically differentiated populations of "gamodemes." Linkage disequilibrium within gamodemes is allowed in two of the models. Maximum likelihood criteria are used to fit the models to the observed numbers of zygotes in a sample. A fitting-testing sequence for choosing a best model is described and the power of the test is analyzed. The statistical characteristics of the genetic parameter estimates were examined by simulation studies. In general, estimates were reliable when allele frequency differences between gamodemes were greater than 0.30 at both loci. This method may be used to study the population structure of samples with fewer heterozygotes than expected for Hardy-Weinberg populations, including the detection and genetic description of sibling species having overlapping ranges.--An example is given for Drosophila longicornis and D. propachuca, two sibling species within the mulleri complex of the repleta group which have been studied in detail using more conventional techniques. The reanalysis using the approach derived in this paper confirmed the reproductive isolation of these two species, and hinted at the possibility of further subdivision within D. propachuca.

Alleles↗
PubMed1979

Functional relationships between the hippocampus and the cerebellum: an electrophysiological study of the cat.

1. Functional interrelationships between the hippocampus and the cerebellum have been investigated in the anaesthetized cat. Plots of the stimulating and recording sites as well as the latency range of the responses indicated the extent of ascending and descending lines of operation between these two structures. 2. Stimulation of the fastigial nucleus evoked the discharge of single hippocampal units on both sides of the brain. Early responses had a mean latency of 12 msec and late responses had a mean latency of 21 msec. Increasing the intensity of the stimulus had little effect on the patterns of discharge. 3. There was no topographical organization within the hippocampus. On the other hand, the activity evoked by a cutaneous stimulus was shown to be greatly depressed by a preceding cerebellar stimulus, particularly at intervals 30--40 msec between the two stimuli. 4. Cerebellar responses evoked by stimulating the hippocampus were found mainly in lobule VI of the posterior lobe. Early and late responses were frequently recorded in the same trace, ipsilateral stimulation yielding the shortest latencies. Increasing the intensity of the stimulus increased the likelihood of there being a response and increased the number of spikes in each discharge. Hippocampal stimulation also had a profound influence on resting cerebellar discharges. 5. Symmetrical points in the two hippocampi were chosen for conditioning and testing sequences. The conditioning stimulus had a long-lasting inhibitory effect on the test response followed by a slow recovery. 6. The location and extent of hippocampal influences on the cerebellum were determined by plotting the presence or absence of a response at each stimulated site. The results indicated the existence of bilateral descending projections containing fast and slow components in conformity with the known conduction properties of mossy fibre and climbing fibre inputs. 7. The physiological significance of interrelationship between the hippocampus and the cerebellum is discussed. It seems that there are many similarities as well as fundamental differences in the cerebellar control of movement under normal circumstances and in conditions of stress.

Animals↗
PubMed1992

Transcription from varicella-zoster virus gene 67 (glycoprotein IV).

Three transcripts map to the varicella-zoster virus (VZV) open reading frame (ORF) 67, which encodes glycoprotein IV (gpIV). All of these transcripts are polyadenylated and are transcribed from left to right towards the genomic terminal short repeats. Previous Northern (RNA) blot analyses suggested that the most abundant of these transcripts (1.65 kb) might code for gpIV. We performed S1 nuclease protection and primer extension assays and determined that the 5' terminus of the 1.65-kb transcript maps 91 bp upstream from the gpIV initiation codon. An AT-rich region (ATAAA), -28 bp from the cap site, is a potential TATA box, and at -71 bp there is a consensus CCAAT box motif. The 3' end of the 1.65-kb transcript is 20 bp downstream of two overlapping polyadenylation signals, AATAAA and ATTAAA, and just downstream of the 3' terminus is a GU-rich sequence. These results are reminiscent of data from our analysis of the VZV gpV gene, confirming that VZV appears able to use unusual TATA box motifs. Many canonical TATA sequences are present upstream from these VZV transcriptional start sites but, apparently, are not used. We tested sequences upstream from the gpIV cap site for promoter activity in transient expression experiments by cloning a DNA fragment (+63 to -343 bp) into pCAT3M, which contains a chloramphenicol acetyltransferase reporter gene. This clone showed little constitutive promoter activity but was activated more than 200-fold by infection with VZV and 5-fold with herpes simplex virus. The two known VZV transactivating genes (those for ORF 4 and ORF 62) were tested for their abilities to activate expression from the gpIV promoter by using their cognate promoters. The ORF 4 gene was minimally active, whereas the ORF 62 gene gave twofold induction; both genes, acting together, gave fivefold induction. However, replacement of the IE62 promoter with the immediate-early cytomegalovirus promoter in the ORF 62 construct gave over 40-fold induction of chloramphenicol acetyltransferase activity under the gpIV promoter in the same assay.

Amino Acid Sequence↗
PubMed1975

Color vision: blue deficiencies in children?

Recent publicized reports based on the use of the Farnsworth Panel D-15 test suggest that a large percentage of young children have a deficiency of blue vision (tritan type). In our study, 413 school children (ages 3 to 10) were tested with both the Farnsworth Panel D-15 test, as well as the A.O. H-R-R plates. None of the children failed either test for blue-yellow vision when traditional scoring instructions were observed. As in previous reports, we find that the children make a number of minor errors which adults rarely make. These errors show marked age-related patterns, being more frequent in younger children. However, further analysis of these errors revealed that the relative frequency with which particular error types were made on the D-15 test was significantly correlated with the existing perceived color difference data for the visually normal adult population. In addition, retesting significantly reduced all error types and reversing the test sequence demonstrated that most of the minor errors were made in the last half of the test regardless of the color vision task. The overall increase in the number of minor test errors seen with young children seems unrelated to color defects. The modified scoring methods in conjunction with the characteristics of the Panel D-15 test design account for the high percentage of errors classified as errors of blue vision.

Age Factors↗
PubMed1987

Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.

Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rab1, -2, -3, and -4, encoding proteins homologous to the yeast YPT protein. Amino acid homology scores with YPT range from 75% for rab1 to 37% for rab4, whereas the homologies with p21 ras and other ras-related proteins are approximately equal to 30%, and homologous residues were clustered in the regions involved in GTP/GDP binding. Another striking similarity shared by the rab and the other ras-related proteins is the conservation of at least one cysteine residue near the carboxyl-terminal end involved in the membrane binding of the ras proteins. rab1 is a mammalian homolog of the yeast YPT gene, and the four rab genes constitute an additional branch of the ras gene superfamily that to our knowledge has not been described in higher eukaryotes.

Amino Acid Sequence↗
PubMed1991

Binding of human glutaminyl-tRNA synthetase to a specific site of its mRNA.

The human glutaminyl-tRNA synthetase is able to bind to its own mRNA. The enzyme contains two binding regions. One is located in the central section of the enzyme which includes its most hydrophilic portion with ten lysine residues in a block of 20 amino acids. This part of the enzyme binds unspecifically to all RNA sequences tested. A second binding region is located in that part of the enzyme which shows high degrees of sequence similarities with the bacterial and yeast glutaminyl-tRNA synthetases, and which is most likely responsible for the charging of tRNA with glutamine. This second RNA binding region specifically interacts with a site in the 3' noncoding region of the synthetase's mRNA. The binding site in the mRNA is characterized by an extended secondary structure that includes elements of the 'identity set' of nucleotides recognized by the enzyme when interacting with tRNA. We discuss possible physiological implications of the interaction between glutaminyl-tRNA synthetase and its mRNA.

Base Sequence↗
PubMed1991

Localization ability in infants with simulated unilateral hearing loss.

This study investigated the feasibility of using a localization task to rule out unilateral hearing loss in infants. Fourcorner localization ability was assessed in 29 normal-hearing infants (9-20 mo) using four different test stimuli. In these same infants, a mild unilateral hearing loss was simulated by occlusion of the external auditory canal and the test sequence was repeated. Analysis of front-back, right-left, and combined errors for each of the test stimuli revealed that this type of task may allow detection of unilateral hearing loss as slight as 25 dB HL.

Acoustic Stimulation↗