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Aluminum (Al) is the most abundant metal in the earth's crust, and humans are exposed to Al through sources like food, cosmetics, and medication. So far, no comprehensive data on the Al distribution between and within human tissues were reported. We measured Al concentrations in 24 different tissue types of 8 autopsied patients using ICP-MS/MS (inductively coupled plasma-tandem mass spectrometry) under cleanroom conditions and found surprisingly high concentrations in both the upper and inferior lobes of the lung and hilar lymph nodes. Al/Si ratios in lung and hilar lymph node samples of 12 additional patients were similar to the ratios reported in urban fine dust. Histological analyses using lumogallion staining showed Al in lung erythrocytes and macrophages, indicating the uptake of airborne Al in the bloodstream. Furthermore, Al was continuously found in PM 2.5 and PM 10 fine dust particles over 7 years in Upper Austria, Austria. According to our findings, air pollution needs to be reconsidered as a major Al source for humans and the environment.

Aluminum is added to many food colors to change their solubility. This study compares the aluminum-containing food color carmine with its aluminum-free version carminic acid (both E 120), hypothesizing that the addition of aluminum does not only change the color's solubility, but also its effects on human cells. We could show that carmine, but not carminic acid, is taken up by gastrointestinal Caco-2 and umbilical vein endothelial cells (HUVEC). Clear differences between gene expression profiles of Caco-2 cells exposed to carmine, carminic acid or control were shown. KEGG analysis revealed that carmine-specific genes suppress oxidative phosphorylation, and showed that this suppression is associated with neurodegenerative diseases such as Alzheimer and Parkinson disease. Furthermore, carmine, but not carminic acid, increased proliferation of Caco-2 cells. Our findings show that a food color containing aluminum induces different cellular effects compared to its aluminum-free form, which is currently not considered in EU legislation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Coronavirus disease 2019 has been reported to be a prothrombotic condition; however, multicenter data comparing this with other viral pneumonias in those requiring extracorporeal membrane oxygenation are lacking. We conducted a multicenter study using whole-body CT to examine the prevalence, severity, and nature of vascular complications in coronavirus disease 2019 in comparison with patients with other viral pneumonias., Design: We analyzed whole-body CT scans for the presence of vascular thrombosis (defined as pulmonary artery thrombus, venous thrombus, systemic arterial thrombus, or end-organ infarct). The severity, distribution, and morphology of pulmonary artery thrombus were characterized. Competing risk cumulative incidence analysis was used to compare survival with discharge., Setting: Three centers of the English national extracorporeal membrane oxygenation service., Patients: Consecutive patients admitted with either coronavirus disease 2019 or noncoronavirus disease 2019 viral pneumonia admitted from January 2019., Interventions: None., Measurements and Main Results: One-hundred thirty-six patients (45.2 ± 10.6 yr old, 39/146 [27%] female) requiring extracorporeal membrane oxygenation support underwent whole-body CT scans at admission. Of these, 86 had coronavirus disease 2019 pneumonia, and 50 had noncoronavirus disease 2019 viral pneumonia. Vascular thrombosis was seen more often in patients with coronavirus disease 2019 (odds ratio, 12.9 [95% CI 4.5-36.8]). In those with coronavirus disease 2019, 57 (73%) demonstrated pulmonary artery thrombus or pulmonary perfusion defects. Eighty-two percent of thrombus exhibited emboli-like morphology. The location of pulmonary artery thrombus and parenchymal perfusion defects was only concordant in 30% of cases. The risk of mortality was higher in those with coronavirus disease 2019 compared with noncoronavirus disease 2019 pneumonia (χ2 = 3.94; p = 0.047). Mortality was no different in coronavirus disease 2019 patients with or without vascular thrombosis (χ2 = 0.44; p = 0.51)., Conclusions: In patients who received extracorporeal membrane oxygenation, coronavirus disease 2019 is associated with a higher prevalence of vascular thrombosis compared with noncoronavirus disease viral pneumonias. The pattern of pulmonary vascular changes suggests concurrent embolic disease and small vessel disease. Despite this, vascular thrombosis was not linked to poorer short-term prognosis in those with coronavirus disease 2019., Competing Interests: Dr. Weir-McCall received support for article research from Research Councils UK. Dr. Toshner received funding from Bayer and Actelion/Jansen; he disclosed he is a member of MorphogenIX scientific advisory board. Drs. Toshner and Jacob received funding from GlaxoSmithKline. Dr. Rodrigues received funding from Sanofi. Drs. Rodrigues and Jacob received funding from National Health Service Digital. Dr. Jacob received funding from Boehringer Ingelheim and Roche; he received support for article research from Wellcome Trust/Charity Open Access Fund. Dr. Jacob is supported by a grant 209553/Z/17/Z from the Wellcome Trust and the National Institute for Health Research University of College London Biomedical Research Center. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.)

Already at the very beginning of the COVID-19 pandemic, an extensive PCR and antigen testing strategy was considered necessary and subsequently also proved successful in order to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on international and national levels. However, equally important will be the continuous monitoring of the seroprevalence status of populations from defined regions to detect-in a timely manner-any recurrence of infections or an eventual decline in antibody levels of vaccinated individuals, especially in the emerging post-pandemic situation. The aim of this study was to estimate the prevalence of SARS-CoV-2-specific immunoglobulin G antibodies in the federal state of Upper Austria (Austria) during the period of December 2020 until April 2021. To achieve this goal, we have analyzed anonymized data on the immune status of self-referral volunteers that have been determined at local pharmacies through a low-entry-barrier point-of-care analysis approach. The seroprevalence values for immunoglobulin type G antibodies against SARS-CoV-2 antigens obtained by rapid diagnostic testing on peripheral blood from volunteers reflect the current population-based estimates reported in the literature as well as the positivity rates detected by PCR-screening analyses. In conclusion, broad-based monitoring of IgG antibodies by means of a point-of-care testing network represents a valuable tool to assess the current immune situation within regionally defined populations., (© 2022. The Author(s).)

Objective: To evaluate the inter- and intraobserver agreement of COVID-RADS and CO-RADS reporting systems among differently experienced radiologists in a population with high estimated prevalence of COVID-19., Methods and Materials: Chest CT scans of patients with clinically-epidemiologically diagnosed COVID-19 were retrieved from an open-source MosMedData data set, randomised, and independently assigned COVID-RADS and CO-RADS grades by an abdominal radiology fellow, thoracic imaging fellow and a consultant cardiothoracic radiologist. The inter- and intraobserver agreement of the two systems were assessed using the Fleiss' and Cohen's κ coefficients, respectively., Results: A total of 200 studies were included in the analysis. Both systems demonstrated moderate interobserver agreement, with κ values of 0.51 [95% confidence interval (CI): 0.46-0.56] and 0.55 (95% CI: 0.50-0.59) for COVID-RADS and CO-RADS, respectively. When COVID-RADS and CO-RADS grades were dichotomised at cut-off values of 2B and 4 to evaluate the agreement between grades representing different levels of clinical suspicion for COVID-19, the interobserver agreement became substantial with κ values of 0.74 (95% CI: 0.66-0.82) for COVID-RADS and 0.73 (95% CI: 0.65-0.81) for CO-RADS. The median intraobserver agreement was considerably higher for CO-RADS reaching 0.81 (95% CI: 0.43-0.76) compared with 0.60 (95% CI: 0.43-0.76) of COVID-RADS., Conclusions: COVID-RADS and CO-RADS showed comparable interobserver agreement, which was moderate when grades were compared head-to-head and substantial when grades were dichotomised to better reflect the underlying levels of suspicion for COVID-19. The median intraobserver agreement of CO-RADS was, however, considerably higher compared with COVID-RADS., Advances in Knowledge: This paper provides a comprehensive review of the newly introduced COVID-19 chest CT reporting systems, which will help radiologists of all sub-specialties and experience levels make an informed decision on which system to use in their own practice., (© 2020 The Authors. Published by the British Institute of Radiology.)

Background: Femoral stems with bimodular (head-neck as well as neck-body) junctions were designed to help surgeons address patients' hip anatomy individually. However, arthroplasty registers have reported higher revision rates in stems with bimodular junctions than in stems with modularity limited to the head-neck trunnion. However, to our knowledge, no epidemiologic study has identified patient-specific risk factors for modular femoral neck fractures, and some stems using these designs still are produced and marketed., Questions/purposes: The purposes of this study were (1) to establish the survival rate free from aseptic loosening of one widely used bimodular THA design; (2) to define the proportion of patients who experienced a fracture of the stem's modular femoral neck; and (3) to determine factors associated with neck fracture., Methods: In this retrospective, nationwide, multicenter study, we reviewed 2767 bimodular Profemur® Z stems from four hospitals in Slovenia with a mean followup of 8 years (range, 3 days to 15 years). Between 2002 and 2015, the four participating hospitals performed 26,132 primary THAs; this implant was used in 2767 of them (11%). The general indications for using this implant were primary osteoarthritis (OA) in 2198 (79%) hips and other indications in 569 (21%) hips. We followed patients from the date of the index operation to the date of death, date of revision, or the end of followup on March 1, 2018. We believe that all revisions would be captured in our sample, except for patients who may have emigrated outside the country, but the proportion of people immigrating to Slovenia is higher than the proportion of those emigrating from it; however, no formal accounting for loss to followup is possible in a study of this design. There were 1438 (52%) stems implanted in female and 1329 (48%) in male patients, respectively. A titanium alloy neck was used in 2489 hips (90%) and a cobalt-chromium neck in 278 (10%) hips. The mean body mass index (BMI) at the time of operation was 29 kg/m (SD ± 5 kg/m). We used Kaplan-Meier analysis to establish survival rates, and we performed a chart review to determine the proportion of patients who experienced femoral neck fractures. A binary logistic regression model that controlled for the potential confounding variables of age, sex, BMI, time since implantation, type of bearing, diagnosis, hospital, neck length, and neck material was used to analyze neck fractures., Results: There were 55 (2%) aseptic stem revisions. Survival rate free from aseptic loosening at 12 years was 97% (95% confidence interval [CI] ± 1%). Fracture of the modular neck occurred in 23 patients (0.83%) with a mean BMI of 29 kg/m (SD ± 4 kg/m.) Twenty patients with neck fractures were males and 19 of 23 fractured necks were long. Time since implantation (odds ratio [OR], 0.55; 95% CI 0.46-0.66; p < 0.001), a long neck (OR, 6.77; 95% CI, 2.1-22.2; p = 0.002), a cobalt-chromium alloy neck (OR, 5.7; 95% CI, 1.6-21.1; p = 0.008), younger age (OR, 0.91; 95% CI, 0.86-0.96; p < 0.001), and male sex (OR, 3.98; 95% CI, 1.04-14.55; p = 0.043) were factors associated with neck fracture., Conclusions: The loosening and neck fracture rates of the Profemur® Z stem were lower than in some of previously published series. However, the use of modular femoral necks in primary THA increases the risk for neck fracture, particularly in young male patients with cobalt-chromium long femoral necks. The bimodular stem we analyzed fractured unacceptably often, especially in younger male patients. For most patients, the risks of using this device outweigh the benefits, and several dozen patients had revisions and complications they would not have had if a different stem had been used., Level of Evidence: Level III, therapeutic study.

The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.

The aims of this study were to determine (1) whether the duration of closed suction drainage affects the occurrence and duration of prolonged serous drainage and (2) if closed suction drains could be omitted according to the wound and/or thigh appearance after unilateral primary total hip arthroplasty. One hundred thirty-nine patients undergoing total hip arthroplasty were randomized into 3 groups: 42 patients received no drainage, 46 patients received drainage for 24 hours, and 51 patients received drainage for 48 hours. No differences with respect to occurrence and duration of prolonged serous drainage were found between the 2 groups with drains. Although no prolonged serous drainage occurred, the swelling of the thigh was significantly greater (P < .001) and the occurrence of prolonged thigh pain was significantly higher (P = .01) in the group without drainage., (2010 Elsevier Inc. All rights reserved.)

Despite intensive research in the field of oral anticoagulants over the last decade, simple and effective long-term prevention of thromboembolism is still an unmet need. In addition to drug discovery approaches, the development of novel oral drug delivery systems (DDSs) of clinically well-established anticoagulants presents an intriguing mean of improvement of anticoagulant therapy. The latter topic is therefore the focus of the present review. All relevant clinical trials with anticoagulants formulated in the oral DDS are reviewed, and selected preclinical examples of promising novel anticoagulant DDSs are also described. For greater understanding, a background on DDS and drug absorption from the gastrointestinal tract is also provided. Three leading approaches for the oral anticoagulant DDS are currently being investigated in clinical settings, all relying on coadministration of anticoagulants with specific carriers. In contrast to the clinical setting, a diverse range of possibilities for oral delivery of anticoagulant are being investigated in preclinical trials (e.g., nanotechnology), and it would be therefore interesting to examine their performance in clinical trials.

Penicillin-binding proteins (PBPs), the main targets of beta-lactam antibiotics, are membrane-associated enzymes that catalyze the two last steps in the biosynthesis of peptidoglycan. In Streptococcus pneumoniae, a major human pathogen, the surge in resistance to such antibiotics is a direct consequence of the proliferation of mosaic PBP-encoding genes, which give rise to proteins containing tens of mutations. PBP2b is a major drug resistance target, and its modification is essential for the development of high levels of resistance to piperacillin. In this work, we have solved the crystal structures of PBP2b from a wild-type pneumococcal strain, as well as from a highly drug-resistant clinical isolate displaying 58 mutations. Although mutations are present throughout the entire PBP structure, those surrounding the active site influence the total charge and the polar character of the region, while those in close proximity to the catalytic nucleophile impart flexibility onto the beta3/beta4 loop area, which encapsulates the cleft. The wealth of structural data on pneumococcal PBPs now underlines the importance of high malleability in active site regions of drug-resistant strains, suggesting that active site "breathing" could be a common mechanism employed by this pathogen to prevent targeting by beta-lactams.

A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5'-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 microM, was discovered.

Cytotoxicity screening of new chemical entities in antibacterial drug discovery discerns between cytotoxic and antimicrobial activity, thus providing predictive evidence for selective toxicity. The objective of this study was to evaluate 3 cytotoxicity assays in identifying novel antibacterial hits with desired safety margins. The endpoints in assays comprised adenylate kinase (AK) release rate as an indicator of membrane rupture (Toxilight), intracellular adenosine triphosphate (CellTiter-Glo), and reduction of resazurin (CellTiter-Blue) both as indicators of cell metabolic activity. In the CellTiter-Glo and the CellTiter-Blue assays, 7 of 8 selected compounds showed cytotoxicity, whereas in the Toxilight assay, 3 of 8 compounds significantly reduced cell viability in the ChoK1 and the JurkatE6.1 cell line. The CellTiter-Glo assay proved to be the most sensitive among the evaluated assays, and excellent Z' values were obtained in the 96-well plate (Z' > 0.83). The CellTiter-Glo assay was clearly superior to the CellTiter-Blue and the Toxilight assay for the initial cytotoxicity screening. Moreover, the application of the CellTiter-Glo assay to determine mammalian cell toxicity versus the antibacterial effect ratio contributed to early identification of antibacterial hits with desired safety margins. The chemical structures of these novel antibacterial hits are disclosed herein.

Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition state analogues, displayed IC50 values ranging from 80 to 600 microM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three-domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C-terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns-length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure-based inhibitor design., (2007 Wiley-Liss, Inc.)

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.

DNA gyrase is an attractive and well established target for the development of antibacterial agents. This bacterial enzyme, whose biological function is to control the topological state of DNA molecules, consists of two catalytic subunits; GyrA is responsible for DNA breakage and reunion, while the subunit GyrB contains the ATP-binding site. Coumarins and cyclothialidines are natural products that inhibit the ATPase activity of DNA gyrase by blocking the binding of ATP to subunit GyrB. The mechanism of action of these compounds was exhaustively characterized by biochemical methods and supported by protein crystallography. The abundance of crystallographic data on the N-terminal domain of GyrB in its complexes with various ligands has enabled the structure-based design of novel efficient chemotypes as inhibitors of the ATPase domain. This review summarizes the discovery of ATPase inhibitors of DNA gyrase B in the last decade and their development as potential antibacterial agents.

The widespread emergence of pathogenic bacterial strains with resistance to antibiotics is becoming a serious threat to public health. Continuous development of novel antibacterials therefore remains one of the biggest challenges to science and unmet needs in the clinics. The biosynthetic pathway of bacterial peptidoglycan, an essential building block of cell walls, has been well studied and appears to be a rich source of attractive enzyme targets for new antibacterials. We have therefore reviewed the intracellular part of peptidoglycan biosynthesis, including the enzymes GlmS, GlmM, GlmU for formation of UDP-GlcNAc, subsequent pentapeptide synthesis by MurA-MurF, and its connection to lipid carrier by MraY and MurG. Naturally occurring inhibitors and the development of low-molecular weight inhibitors of the intracellular part of peptidoglycan synthesis are presented.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) modulate the biological potency of estrogens and androgens by interconversion of inactive 17-keto-steroids and their active 17beta-hydroxy- counterparts. We have shown previously that flavonoids are potentially useful lead compounds for developing inhibitors of 17beta-HSDs. In this paper, we describe the synthesis and biochemical evaluation of structurally analogous inhibitors, the trans-cinnamic acid esters and related compounds. Additionally, quantitative structure-activity relationship (QSAR) and modelling studies were performed to rationalize the results and to suggest further optimization. The results stress the importance of a hydrogen bond with Asn154 and hydrophobic interactions with the aromatic side chain of Tyr212 for optimal molecular recognition.

Fighting bacterial resistance is a challenging task in the field of medicinal chemistry. DNA gyrase represents a validated antibacterial target and has drawn much interest in recent years. By a structure-based approach we have previously discovered compound 1, an indolinone derivative, possessing inhibitory activity against DNA gyrase. In the present paper, a detailed biophysical characterization of this inhibitor is described. Using mass spectrometry, NMR spectroscopy, and fluorescence experiments we have demonstrated that compound 1 binds reversibly to the ATP-binding site of the 24 kDa N-terminal fragment of DNA gyrase B from Escherichia coli (GyrB24) with low micromolar affinity. Based on these data, a plausible molecular model of compound 1 in the active site of GyrB24 was constructed. The predicted binding mode explains the competitive inhibitory mechanism with respect to ATP and forms a useful basis for further development of potent DNA gyrase inhibitors.

We describe here the fragment-based design of potent DNA gyrase inhibitors. Using the tools of virtual screening and NMR spectroscopy we identified the binding of two low-molecular weight fragments (2-aminobenzimidazole and indolin-2-one) to the 24kDa N-terminal fragment of DNA gyrase B. Further in silico optimization of indolin-2-one led to the discovery of potent DNA gyrase inhibitors.

In the present work we explore the possibility of an in-depth computational analysis of available experimental X-ray structures in the specific case of a series of alpha-thrombin and trypsin complexes with their respective inhibitors for the development of a novel scoring function based on molecular electrostatic potential computed at the contact surface in the enzyme-inhibitor molecular complex. We subsequently employ the chemometrical approach to determine which are the interactions in the large volume of data that determine the resulting experimental binding constant between ligand and receptor. The results of the model evaluated with molecules in the independent validation set show that a reasonable average error of 1.30 log units of the difference between experimental and calculated binding constants was achieved in the system thrombin-trypsin, which is comparable with those of methods from the literature. Furthermore, by a careful preparation of the Kohonen top layer in the artificial neural network approach that is normally perceived as a "black box device", we have been able to follow the implications of the structure of the inhibitor-enzyme complex for the inhibitor's binding constant. The method appears to be suitable for evaluation of selectivity in structurally similar enzymatic systems, which is currently an important problem in drug design., (Copyright 2004 American Chemical Society)

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

Potato tubers contain considerable amounts of inhibitors of cysteine proteinases. The majority of inhibitory activity is due to low-molecular mass inhibitors differing in isoelectric points. Three of them were obtained in homogenous form, namely PCPIs (potato cysteine proteinase inhibitors) 6.6 (Mr 25,000), 8.3 and 9.4 (both Mr 22,000). They all appeared to be single-chain proteins. The amino-acid composition and the N-terminal amino-acid sequences show that at least two of them are homologous proteins, but so far no homology to the inhibitors of the cystatin super-family or to any other sequenced potato proteins is apparent. PCPIs inhibit papain and human cathepsins B, H, and L. The inhibitors interact with enzymes in apparently equimolar fashion, the interaction is of the tight binding type with Ki values ranging from 10(-6)M to 10(-11)M.

The cathepsins B, H and L of human origin were isolated in pure form in sufficient quantities for structural characterization. The complete amino acid sequence of human liver cathepsin B was determined. Partial amino acid sequences of the human kidney cathepsin H and L show the highly conserved region around the active site cysteine. The cysteine proteinase inhibitors stefin A, human stefin B and human cystatin C were isolated, characterized and sequenced. Their amino acid sequences are compared with sequences of other protein inhibitors of the stefin and cystatin family, showing a high degree of homology throughout both families. The stefin and cystatin family, together with newly discovered kininogen family belong to the same superfamily of cystatins. The constructed dendrogram shows that the most closely related inhibitors so far sequenced are human stefin B and rat liver TPI.