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PubMed1992

Primary structure of a Thomsen-Friedenreich-antigen-specific lectin, jacalin [Artocarpus integrifolia (jack fruit) agglutinin]. Evidence for the presence of an internal repeat.

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

Amino Acid Sequence↗

PubMed1986

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation.

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.

Amino Acid Sequence↗

PubMed1992

Cloning and developmental expression of the alpha 3 chain of chicken type IX collagen.

Fibrous and nonfibrous collagens comprise two major groups within the collagen family and both groups are found in a diverse variety of tissue fabrics. Type IX collagen is in the nonfibrous group; three different subunits of type IX collagen have been identified and the alpha 1 and alpha 2 subunits have been cloned. Using molecular cloning methods we have isolated, from an embryonic chicken cartilage library, cDNA clones which code for the entire alpha 3 chain of chicken type IX collagen. The cDNA clones encompass 2416 base pairs which have a conceptual open reading frame for a protein containing 675 amino acids including 193 Gly-X-Y repeats. These collagen repeats are in three separate domains which are interspersed with four major noncollagen domains. The collagen repeats also have four minor interruptions. This chain organization directly aligns with both the alpha 1 and alpha 2 chains of chicken type IX collagen. Comparison of the deduced amino acid sequence with peptide sequences of type IX collagens shows identity with 95 of the 96 known residues of the chicken alpha 3 chain and 81 of the 98 known residues of the bovine alpha 3 chain. The identical residues match those in five peptide fragments, two from the bovine protein and three from the chicken protein. The chicken and bovine alpha 3 chains have conserved cross-linking sites, separated by 137 residues which span 40 nm, the length of the hole zone in a collagen fibril. The NC3 domain of the chicken alpha 3 chain contains a repeat Cys-Pro motif which is present in both vertebrate and invertebrate nonfibrillar collagens. Northern blot hybridization exhibits a major mRNA of about 3.3 kilobases; this transcript is found in cartilaginous tissues in the embryo, including the developing limb and is not detected in other tissues or in the precondensation stage of limb development. The composite data delineate the primary structure of the alpha 3 chain of chicken type IX collagen, show its close relationship to the alpha 1 and alpha 2 chains, demonstrate its mRNA transcript, and show the appearance of that transcript in tissues of the developing chick embryo.

Amino Acid Sequence↗

PubMed2025

Hierarchical integration of mNGS, PCR, and other conventional methods for precision TB diagnostics.

UNLABELLED: This study systematically compared the diagnostic accuracy of seven assays for detecting the Mycobacterium tuberculosis complex, including metagenomic next-generation sequencing (mNGS), droplet digital polymerase chain reaction, real-time quantitative polymerase chain reaction, EasyNAT MTC, GeneXpert MTB/RIF, interferon-gamma release assay (IGRA), and acid‒fast staining (AFS). We try to select appropriate combinations of tuberculosis (TB) detection methods for regions with varying levels of medical resources, based on sensitivity, cost-effectiveness, and operational feasibility. A retrospective analysis was conducted on 141 samples collected from patients with suspected active TB at The First Affiliated Hospital of Sun Yat-sen University between April 2022 and April 2024. Among these samples, there were 100 cases assigned to the case group and 41 cases to the control group, based on the tuberculosis diagnostic criteria. Historical data for Xpert, IGRA, and AFS were collected, and parallel experiments using mNGS, droplet digital PCR (ddPCR), real-time quantitative polymerase chain reaction (RT-qPCR), and EasyNAT were conducted on all samples. Diagnostic performance was evaluated by comparing it with the final clinical diagnoses. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic (ROC) curve analysis were conducted, along with DeLong tests for statistical comparison. Compared with the final clinical diagnosis, mNGS demonstrated the highest sensitivity (100%), followed by IGRA (79.2%), EasyNAT (79.1%), RT-qPCR (78.0%), ddPCR (75.8%), Xpert (75.3%), and AFS (16.7%). The specificity was 100% for both Xpert and AFS, followed by ddPCR (97.6%), RT-qPCR (95.1%), EasyNAT (92.7%), IGRA (72.7%), and mNGS (75.6%). ROC analysis revealed a significantly greater area under the ROC curve for mNGS (0.878) than for ddPCR (0.817, P = 0.031). DeLong tests revealed statistically significant differences in diagnostic performance between mNGS and ddPCR (P < 0.05) and between IGRA and AFS (P < 0.01). mNGS uniquely identified the pathogens involved in co-infection and quantified pathogen-specific sequencing reads. Through a comprehensive evaluation of the diagnostic efficacy, cost-effectiveness, and timeliness of tuberculosis detection methods, we propose corresponding combinations of TB testing approaches for regions with different healthcare resources. For undeveloped regions with limited resources, a combination of AFS +EasyNAT + chest X-ray is recommended. Primary care facilities may additionally employ IGRA + RT-qPCR. Intermediate-level hospitals can incorporate Xpert MTB/RIF for drug resistance testing, while tertiary hospitals or specialized centers should, on the basis of these fundamental tests, utilize mNGS for diagnosis and ddPCR for therapeutic monitoring in patients with complex mixed infections. IMPORTANCE: This study is the first to comprehensively evaluate the diagnostic efficacy, cost-effectiveness, and timeliness of seven TB detection methods in a single-center cohort. Our findings provide actionable solutions for optimizing TB diagnostics in diverse healthcare ecosystems, aligning with the WHO's End TB Strategy to ensure equitable access to rapid diagnostics.

Humans↗

PubMed1980

Determination of polypeptide amino acid sequences from the carboxyl terminus using angiotensin I converting enzyme.

A method for sequence analysis of polypeptides starting at the carboxyl terminus is described that utilizes degradation of the polypeptide into dipeptides with angiotensin I converting enzyme. Dipeptides were identified by gas chromatography-mass spectroscopy. Dipeptide alignment was achieved by replicate digestion of the polypeptide after modification at the carboxyl terminus either by chemical or enzymatic removal of one residue or by addition of a single residue. The addition reaction involved coupling of L-alpha-aminobutyric acid under conditions described herein which yielded essentially complete conversions. Unlike sequence determination methods that commence from the polypeptide amino terminus, this procedure does not require that a polypeptide have a free amino terminus for successful application. A number of polypeptides with varying chain lengths (up to 49 residues), containing among them most of the common amino acids, have been successfully analyzed in amounts as low as 5 nmol.

Amino Acid Sequence↗

PubMed1989

Crystallographic refinement of interleukin 1 beta at 2.0 A resolution.

The structure of human recombinant interleukin 1 beta (IL-1 beta) has been refined by a restrained least-squares method to a crystallographic R factor of 17.2% to 2.0 A resolution. One-hundred sixty-eight solvent molecules have been located, and isotropic temperature factors for each atom have been refined. The overall structure is composed of 12 beta-strands that can best be described as forming the four triangular faces of a tetrahedron with hydrogen bonding resembling normal antiparallel beta-sheets only at the vertices. The interior of this tetrahedron is filled by hydrophobic side chains. Analysis of sequence alignments with IL-1 beta from other mammalian species shows the interior to be very well conserved with the exterior residues markedly less so. There does not appear to be a clustering of invariant amino acid side chains on the surface of the molecule, suggesting an area of interaction with the IL-1 receptor. Comparison of the IL-1 beta structure with IL-1 alpha sequences indicates that IL-1 alpha probably has a similar overall folding as IL-1 beta but binds to the receptor in a different fashion. The three-dimensional structure of the IL-1 beta is analyzed in light of what has been suggested by previously published work on mutants and fragments of the molecule.

Amino Acid Sequence↗

PubMed1992

A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives.

Vertebrate fibrinogen is a complex multidomained protein, the structure of which has been inferred mainly from electron microscopy and amino acid sequence studies. Among its most prominent features are two terminal globules, moieties that are mostly composed of the carboxyl-terminal two-thirds of the beta and gamma chains. Sequences homologous to the latter segments are found in several other animal proteins, always as the carboxyl-terminal contributions. An alignment of 15 amino acid sequences from various fibrinogens and related proteins has been used to make judgments about secondary structure. The nature of amino acids at each position in the alignment was used to distinguish alpha helices and beta structure on the one hand from loops and turns on the other, and the resulting assignments compared with predictions of secondary structure by other methods. Additionally, constraints imposed by the locations of cystines, carbohydrate attachment residues, and proteinase-sensitive points provided further insights into the general organization of the postulated secondary structures. Other ancillary data, including the effects of bound calcium and the locations of labeled or variant residues, were also considered. An intriguing similarity to a portion of the recently reported structure of a calcium-dependent lectin is noted.

Amino Acid Sequence↗

PubMed1981

[Human embryonic haemoglobins. The primary structure of the zeta chains (author's transl)].

The primary structure of the embryonic zeta-chains of humans is given. Blood was obtained from a case with hydrops foetalis syndrom due to homozygous alpha-thalassemia 1. The zeta-chains were isolated by high performance liquid chromatography on reversed phase (RP8). The peptides for sequence work were generated by chemical methods (cyanogen bromide cleavage and acid cleavage at the Asp-Pro bond) and enzymatic cleavages with trypsin of unmodified and succinylated zeta-chains. The peptides were separated by high performance liquid chromatography and sequenced by automatic N-terminal degradation procedures. The N-terminal residue of the zeta-chains is blocked. Therefore the sequence of the N-terminal tryptic peptide was determined after incubation with chymotrypsin. The zeta-chains are alpha-type chains and consist of 141 amino acid residues. The alignment of the zeta-chains with the human alpha-chains shows 57 amino acid exchanges: Thus it is evident that there is a greater phylogenetic distance between the alpha type chains than between the beta-type chains.

Amino Acid Sequence↗

PubMed2026

BaGGLS: a Bayesian shrinkage framework for interpretable modeling of interactions in high-dimensional biological data.

MOTIVATION: Biological data is often high dimensional, noisy, and governed by complex interactions among sparse signals. This poses major challenges for interpretability and reliable feature selection. Tasks such as identifying motif interactions in genomics exemplify these difficulties, as only a small subset of biologically relevant features (e.g. motifs) are typically active, and their effects are often non-linear and context-dependent. While statistical approaches often result in more interpretable models, deep learning models have proven effective in modeling complex interactions and prediction accuracy, yet their black-box nature limits interpretability. RESULTS: We introduce BaGGLS, a flexible and interpretable probabilistic binary regression model designed for high-dimensional biological inference involving feature interactions. BaGGLS incorporates a Bayesian group global-local shrinkage prior, aligned with the group structure introduced by interaction terms. This prior encourages sparsity while retaining interpretability, helping to isolate meaningful signals and suppress noise. To enable scalable inference, we employ a partially factorized variational approximation that captures posterior skewness and supports efficient learning even in large feature spaces. In extensive simulations, we compare BaGGLS to frequentist probit regressions (unconstrained and with L1-penalty) as well as a probit model with Markov Chain Monte Carlo (MCMC) sampling under a horseshoe prior. We can show that BaGGLS outperforms the other methods with regard to interaction detection and is many times faster than MCMC sampling under the horseshoe prior. We also demonstrate the usefulness of BaGGLS in the context of interaction discovery from motif scanner outputs (e.g. Find Individual Motif Occurrences (FIMO)) and noisy attribution scores from deep learning models. This shows that BaGGLS is a promising approach for uncovering biologically relevant interaction patterns, with potential applicability across a range of high-dimensional tasks in computational biology. AVAILABILITY: Code is available at gitlab.com/dacs-hpi/baggls.

Bayes Theorem↗

PubMed1987

Genomic organization of the mouse T-cell receptor beta-chain gene family.

We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments.

Animals↗

PubMed1991

Detection and identification of mycoplasmas by amplification of rDNA.

Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.

Base Sequence↗

PubMed1990

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.

Birefringence↗

PubMed1991

S-S bridges of cathepsin B and H from bovine spleen: a basis for cathepsin B model building and possible functional implications for discrimination between exo- and endopeptidase activities among cathepsins B, H and L.

Bovine spleen cathepsin B contains 7 disulfide bridges. Using different chemical and enzymatic cleavage methods we isolated fragments representing the individual disulfides: Cys14-Cys43, Cys26-Cys71, Cys62-Cys128, Cys63-Cys67, Cys100-Cys132, Cys108-Cys119, and Cys148-Cys252. A similar line of approach was applied to determine the S-S bridges of bovine spleen cathepsin H: Cys23-Cys66, Cys57-Cys99, Cys157-Cys207, and Cys212-Cys5A, where Cys5A is located in the propart portion of the procathepsin H chain. On the basis of the knowledge of the S-S bridges of cathepsin B a novel sequence alignment of papain and cathepsin B has been proposed. This enabled us to construct a reasonable 3D-model of cathepsin B and propose the region (a 18 residue insertion between Glu89 and Gly90 of papain) responsible for the carboxypeptidase activity of cathepsin B functioning as a "closure". A similar approach was applied to explain the aminopeptidase activity of cathepsin H. A general model of steric regulation of accessibility of the preformed "endopeptidase-like" binding cleft by distant parts of the polypeptide chain of the proteinases discussed is proposed as a factor determining the mode of binding and thus cleavage of polypeptide substrates.

Amino Acid Sequence↗

PubMed1988

Improvements in a secondary structure prediction method based on a search for local sequence homologies and its use as a model building tool.

This report describes an optimised version of a secondary structure prediction method based on local homologies, using a new data base. A 63% prediction accuracy, for three states, was obtained after elimination of the protein to be predicted and all proteins with a percentage identity greater than 22% from the data base. This corresponds to a 5% increase in accuracy on the original method (Levin et al. FEBS Lett. 205 (1986) 303-308). The flexibility of the method to the incorporation of information extraneous to the prediction was demonstrated by the prediction of the homologous proteins in the data base. Using the percentage identity with the protein to be predicted, to weight the relative importance of each protein, for all proteins with a percentage identity greater than 30%, the mean correct prediction per chain was 87%. As a result this algorithm can be used during the molecular modelling process, both to give an idea of the structural similarity between two proteins and as an aid in the determination of the best alignment. Incorporation of the result of a protein folding type assignment based on the global amino-acid composition increased the overall prediction to 66%.

Algorithms↗

PubMed1992

[DNA diagnosis for inherited metabolic disorders].

Progress in molecular genetics has made it possible to detect the structure of cDNA and genomic DNA of enzyme and protein, as well as the mutation in DNA level. It also provides a vast amount of new information for diagnosis and treatment, and it can actually be used in prenatal diagnosis and carrier screening. There is great hope that patients with genetic disorders can be treated by somatic gene replacement. We introduced here the methods for detection of unknown mutations of inherited metabolic disorders, and screening patients for characterized mutations, and the expression analysis of mutant cDNA, as an example of phenylketonuria.

Amino Acid Metabolism, Inborn Errors↗

PubMed1990

Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. I. Computational analysis.

The tendency for relatively short (less than 500 base-pair) DNA molecules to circularize in the presence of DNA ligase is a sensitive function of both the lateral and torsional flexibilities of the molecules being studied. This tendency is reflected in a quantity known as the j-factor, which is determined experimentally by measuring the relative rates of circle and linear dimer formation at a specified concentration of linear monomer. Shimada & Yamakawa have provided an analytical representation of j that takes account of DNA molecules whose ends are not torsionally aligned. Their approach, however, assumes that contributions from helix writhe are small. Using a Monte Carlo approach for the determination of j, thereby avoiding any assumptions regarding writhe, we demonstrate that the computed, torsion angle-averaged quantity, [j], is exactly reproduced by the corresponding Shimada & Yamakawa quantity for all lengths examined. However, for DNA molecules having lengths that are substantially greater than the persistence length, P, the analysis of experimental ring-closure data using j (Shimada & Yamakawa) may lead to underestimates for the torsional elastic constant C. We demonstrate that no single set of values for P, C and the helical repeat (hR) can produce a reasonable fit of the computed j curve to the experimental values of Shore et al. This observation suggests that P, C and/or hR vary within the set of DNA molecules studied by those authors. The current computational analysis considers the effects on j of single or multiple bends in the helix axis. For single, centrally located bends, the shift in the distribution of end-to-end separations to smaller values is nearly offset by the less favorable polar alignment of the ends of the chain; the net effect being a modest change in j that is not a monotonic function of the bend angle. In contrast, polar alignment, and hence j, can be enhanced dramatically for molecules containing multiple, phased bends. However, for studies of the distribution of circle sizes formed from ligation of bend-containing DNA oligomers, the DNA lengths giving rise to maximal j values are smaller than predicted on the basis of the number of bends and the per-bend angle. This last result suggests that such studies may yield apparent bend angles that are too large.

Chemical Phenomena↗

PubMed1991

[Systematics, differentiation, and detection of bacterial infections-- the family Mycobacteriaceae].

Comparative 16S rRNA sequencing allows to infer natural relationships among bacteria, to characterize and identify microorganisms at a molecular level and to develop DNA probes specific at any desired taxonomic level (e.g. family, genus, species). Probes targeted at ribosomal RNA are suitable for in situ hybridization of whole, intact bacterial cells as well as in polymerase-chain-reaction techniques for sensitive detection and identification of bacteria. Comparative 16S rRNA sequencing provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. Certain growth characteristics, i.e. thermotolerance and growth rate correspond to natural relationships among the mycobacteria. However, the phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotochromogenic and nonphotochromogenic mycobacteria. The use of oligonucleotides targeted at highly or semi-conserved regions within the 16S rRNA molecule allows a universal procedure for amplification and rapid sequence determination of 16S rDNA-gene fragments from any virtually bacterial organism. This method of amplification of 16S rDNA-gene fragments was used to identify a novel, uncultured pathogen and opens new perspectives for other infectious diseases of unknown cause.

Base Sequence↗

PubMed1991

Sequence of 18S rDNA of actinorhizal Alnus glutinosa (Betulaceae).

The small subunit ribosomal DNA for a woody actinorhizal, Alnus glutinosa, was isolated by the PCR method. Amplification products were cloned into the Bluescript SK- vector. Full sequence, 1698 bp, was obtained with NS1 to NS8 primers. Sequence alignments were made by UWGCG sequence data analysis computer programs. 18S rDNA sequence of A. glutinosa was compared to analogous segments of four other angiosperms, tomato, rice, maize and soybean. Sequence homologies are discussed and application for the technique is suggested.

Base Sequence↗