Biomedical subjects

Thomas Ludwig

Publications and source records attributed to Thomas Ludwig.

At least 37 records · Page 2Linked to original sources

PubMed2004

Molecular structure and interaction of recombinant human type XVI collagen.

Collagen XVI is a minor component of at least two different extracellular fibrillar networks of specialized regions of skin and cartilage. In skin, collagen XVI is integrated into particular fibrillin-rich microfibrils lacking an amorphous elastin core. In cartilage, collagen XVI is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Here, we present the first direct evidence for the molecular structure and functional properties of these fibril-associated collagens with interrupted triple helices (FACIT). We have expressed recombinantly the full-length alpha1 chain of human collagen XVI in HEK 293 EBNA cells in large quantities using an episomal expression system. Secreted full-length recombinant collagen XVI forms stable disulfide-bonded homotrimers and is rapidly proteolytically processed to distinct fragments at specific protease sequence motifs, one resembling an aggrecanase recognition site. Limited trypsin digestion assays and thermal transition curves imply sequential thermal denaturation of individual triple helical domains of this recombinant collagen, similar to authentic collagen XVI. Molecular images of collagen XVI reveal rod-like molecules which harbor multiple sharp kinks attributing a highly flexible structure presumably introduced by non-collagenous (NC) regions. Terminally located cloverleaf-shaped nodules correspond to the large NC NC11 domain of trimeric collagen XVI. The total length of individual trimeric recombinant collagen XVI molecules constitutes about 240 nm as calculated by atomic force and negative staining electron microscopy. Recombinant collagen XVI interacts with fibrillin-1 and with fibronectin indicating multiple molecular interactions in which this ubiquitously expressed and versatile FACIT-collagen can participate. In vitro generated collagen XVI provides an indispensable tool for future determination of its function during supramolecular assembly of matrix aggregates and its role in maintenance, organization and interaction of fibrillar structures.

Amino Acid Sequence↗

PubMed2004

Human endothelium: target for aldosterone.

Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies demonstrated the presence of these receptors in nonclassical locations, including the cardiovascular system. We tested the hypothesis whether endothelial cells respond to aldosterone with changes in cell volume, a measure for ion-mediated water movement across the cell membrane. By means of atomic force microscopy in fluid, we measured volume of adherent human umbilical venous endothelial cells exposed for 72 hours to 10 nmol/L aldosterone. Over this period of time, cells swell by approximately 18%. Aldosterone-induced swelling is prevented by 100 nmol/L of the mineralocorticoid receptor antagonist spironolactone, added to the primary endothelial cell culture. Aldosterone-treated cells dramatically shrink when 1 micromol/L of the diuretic amiloride is applied. Cells deprived of aldosterone do not respond to amiloride. Our conclusions are: (1) aldosterone leads to sustained cell swelling inhibited by administration of spironolactone or the sodium channel blocker amiloride; (2) cells respond to amiloride after aldosterone exposure; (3) renal diuretics act on endothelial cells; and (4) both amiloride and spironolactone could be useful for medical applications to prevent aldosterone-mediated endothelial dysfunction.

Aldosterone↗

PubMed2004

'Designer' tumors in mice.

We have developed and tested successfully a general method based on Cre-mediated recombination that can be used for ubiquitous or tissue-specific expression of protein products, including tumor-inducing oncoproteins. Depending on the specificity of a chosen promoter driving cre expression, tumors develop by design in bitransgenic mouse progeny derived by crossing Cre-producing mice with partners carrying a dormant oncogenic transgene (targeted into the 3' noncoding region of the cytoplasmic beta-actin locus) that becomes functional after excision of a 'floxed' DNA segment. To provide proof-of-principle, we have used as models transgenes encoding the polyomavirus middle T antigen (PVMT) and the T antigens of the SV40 early region (SVER). Cre-dependent activation of widespread SVER expression resulted in hyperplasias or invasive tumors affecting particular visceral smooth muscles, whereas Cre-dependent, mammary gland-specific expression of PVMT-induced adenocarcinomas, according to plan. Unexpectedly, we also encountered spontaneous (Cre-independent) oncogene expression occurring as a rare event, which simulates the initiation of sporadic tumors and leads to PVMT-induced hemangiomas and mammary carcinomas or SVER-induced disseminated sarcomas, thus, revealing particular tissue susceptibilities to the actions of these oncoproteins.

Actins↗

PubMed2004

ARB: a software environment for sequence data.

The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.

Data Display↗

PubMed2004

Glioblastoma cells release factors that disrupt blood-brain barrier features.

The blood-brain barrier (BBB), mediated by endothelial tight junctions, is defective in malignant gliomas such as glioblastoma, resulting in cerebral edema and contrast enhancement upon neuroradiological examination. The mechanisms underlying BBB breakdown are essentially unknown. Since non-neoplastic astrocytes are required to induce BBB features of cerebral endothelial cells, it is conceivable that malignant astrocytes have lost this ability due to dedifferentiation. Alternatively, glioma cells might actively degrade previously intact BBB tight junctions. To examine the latter hypothesis, we have employed a transepithelial electrical resistance breakdown assay using monolayers of the C7 subclone of Madin-Darby canine kidney (MDCK-C7) cells forming tight junctions similar to those of BBB endothelial cells. We found that glioblastoma primary cells co-cultured with the MDCK-C7 monolayer (without direct contact of the two cell types) resulted in marked breakdown of electrical resistance, whereas primary cultures derived from low-grade gliomas (fibrillary astrocytoma, oligoastrocytoma) showed delayed or no effects. These results suggest that malignant gliomas have acquired the ability to actively degrade tight junctions by secreting soluble factors, eventually leading to BBB disruption within invaded brain tissue.

Animals↗

PubMed2004

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Lepr(db/db) mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP /frt and CRE /loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele ( Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Lepr(db/db) mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Lepr(flox/flox) mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprDelta17/Delta17 mice, which is indistinguishable from Lepr(neo/neo) and Lepr(db/db) mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Lepr(neo/neo) mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Alleles↗

PubMed2004

Evaluation of head and neck squamous cell carcinoma invasiveness by the electrical resistance breakdown assay.

Invasion of tumor cells into the surrounding tissue is a hallmark of cancer. Squamous cell carcinomas of the head and neck region (HNSCC) are characterized by their early primarily lymphatic metastatic spread. The aim of the present study was to evaluate the use of the electrical resistance breakdown assay for determining HNSCC tumor cell invasiveness. The assay utilizes the high transepithelial electrical resistance (TEER) of an epithelial MDCK-C7 monolayer as a sensitive indicator of monolayer integrity and permeability. MDCK-C7 cells were grown to confluence in microfilter membrane cups. 3 x 10(6) cancer cells of cell lines UM-SCC-3, UM-SCC-27, UMB-SCC-745, UMB-SCC-864, UMB-SCC-969 and UT-SCC-26A derived from HNSCC tumors, were seeded on top of this epithelial test barrier. A7-melanoma cells served as a positive control whereas MDCK-C7 cells were used as a negative control and were applied in the same number as the tested tumor cells. TEER was measured over the following days and compared to control values. A significant reduction in TEER was observed in the UMB-SCC-745, UMB-SCC-969 and UT-SCC-26A cell lines within the first 72 h, whereas no significant reduction in TEER was seen in the UM-SCC-3, UM-SCC-27 and UMB-SCC-864 cell lines. HNSCC cell lines in general are found to be less invasive in the resistance breakdown assay compared to other tumor cells such as A7-melanoma cells, however, the electrical resistance breakdown assay appears capable of demonstrating differences in invasiveness between different HNSCC cell lines and therefore potentially could serve as a versatile tool in distinguishing high and low invasive tumors with a potential application as a diagnostic and prognostic marker in clinical investigations.

Animals↗

PubMed2004

A mouse model of uterine leiomyosarcoma.

We are using an approach that is based on the cre/loxP recombination process and involves a binary system of Cre-producing and Cre-responding transgenic mice to achieve ubiquitous or tissue-specific expression of oncoproteins. To develop mouse models of tumorigenesis, Cre-producers are mated with responder animals carrying a dormant oncogene targeted into the 3' untranslated region of the locus encoding cytoplasmic beta-actin (actin cassette). Production of oncoprotein from a bicistronic message is accomplished in bitransgenic progeny by Cre-mediated excision of a segment flanked by loxP sites that is located upstream from the oncogenic sequence. Widespread Cre-dependent activation and expression of an actin-cassette transgene encoding the T antigens of the SV40 early region (SVER) commencing in embryos was compatible with normal development and did not impair viability. However, at approximately 3 months of age, all female animals developed massive uterine leiomyosarcomas, whereas practically all males exhibited enormously enlarged seminal vesicles because of pronounced hyperplasia of the smooth muscle layers. In addition, because of smooth muscle hyperproliferation, marked dilation of the gallbladder was observed in mice of both sexes. To begin exploring aberrant signaling events in the SVER-triggered tumorigenic pathways, we analyzed the expression profile of leiomyosarcomas by DNA microarray analysis.

Actins↗

PubMed2004

Nephrotoxicity of platinum complexes is related to basolateral organic cation transport.

BACKGROUND: Cisplatin and its analogs oxaliplatin and carboplatin are widely used antitumor drugs. Nephrotoxicity is a common and relevant adverse effect that occurs especially in cisplatin therapy. Cellular and molecular mechanisms of cisplatin-induced nephrotoxicity are not completely understood. The nephrotoxicity of platinum complexes was evaluated by a new in vitro system that utilizes the high Trans Epithelial Electrical Resistance (TEER) of the C7 clone of the MDCK (Madin-Darby canine kidney) cells. By means of this assay system we addressed the question whether the side of application of renal epithelia influences platinum complex toxicity. METHODS: C7 cells were grown in membrane filter cups, and the apical or basolateral membranes were exposed to 100-micromol/L cis-, oxali-, or carboplatin. TEER and caspase-3 activity were determined. Cimetidine was used as an inhibitor of organic cation transporters (OCTs). C7 cell lysates were analyzed for OCT-1 and -2 by Western blot analysis. RESULTS: TEER dropped by 89.5 +/- 9.3% (mean +/- SEM; N= 6) within 24 hours after addition of cisplatin to the basolateral side of C7 cells, while caspase activity increased up to 840.6 +/- 17.4% (mean +/- SEM; N= 6) compared to control cells. Exposure of the apical membrane to cisplatin reduced TEER by only 13.4 +/- 8.7% (mean +/- SEM; N= 6), and increased caspase-3 activity up to 213.9 +/- 7.6% (mean +/- SEM; N= 6). Oxaliplatin and carboplatin reduced TEER to a lesser extent than cisplatin. Oxaliplatin lowered TEER stronger than carboplatin. In general, basolateral application led to higher caspase activities and lower TEERs. The OCT-inhibitor cimetidine inhibited the TEER decrease induced by platinum complexes. Immunoblotting confirmed the presence of OCT-2 in C7 cells. CONCLUSION: Toxic effects of platinum complexes on renal epithelia depend on the platinum complex used and the site of application. We conclude that cell polarity and basolateral transport mechanisms are essential in nephrotoxicity of platinum drugs.

Animals↗

PubMed2004

Platinum complex cytotoxicity tested by the electrical resistance breakdown assay.

The electrical resistance breakdown assay provides a novel approach for the quantification of cytotoxic activity of platinum based anticancer drugs. It is a functional assay system for cancer cell invasion that detects nanoscale alterations of an epithelial test barrier prior to microscopic morphometric changes. We measured changes in transepithelial electrical resistance (TEER) of a tight epithelial MDCK-C7 monolayer in response to highly invasive amelanotic melanoma cells (A7-clone) in combination with different platinum complexes (cis-, oxali- and carboplatin). The efficiency of the electrical resistance breakdown assay was compared a standard method for measurement of cytostatic activity, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT-assay utilizes mitochondrial enzymatic activity to draw conclusions from a functional cell metabolism to the number of living cells in a sample. When human melanoma cells were seeded on top of an electrically tight MDCK-C7 monolayer, electrical leakage occurred within 48 h of co-culture. Electrical resistance breakdown was effectively prevented by cisplatin and its analogs (no significant difference between 100 microM cisplatin and corresponding controls with non-invasive cells). The results of the electrical resistance breakdown and MTT-assay were linearly dependent. Significance of both tests was equivalent, but the electrical resistance breakdown assay gave additional functional information. Compared to oxali- and carboplatin, cisplatin was more effective in preventing TEER-breakdown than reducing the number of tumor cells, giving rise to the assumption that cisplatin can reduce tumor cell number as well as invasiveness. In conclusion the electrical resistance breakdown assay provides a sensitive, continuous and cell-based assay system for the quantification of cancer cell invasiveness and evaluation of chemotherapeutics under physiological conditions.

Animals↗

PubMed2004

Platinum complex toxicity in cultured renal epithelia.

BACKGROUND: Cisplatin is a potent antitumor drug but its clinical use is limited by nephrotoxic side effects. We have found recently, that nephrotoxicity of platinum complexes is related to basolateral organic cation transport. In this study, effects of cell culture conditions on platinum complex toxicity and organic cation transport were investigated by an in vitro system that utilizes the high TransEpithelial Electrical Resistance (TEER) of the C7-clone of the MDCK-(Madin-Darby-Canine-Kidney) cells. METHODS: TEER and caspase-3 activity of cells in microfilter membrane cups were measured after exposure of apical or basolateral membranes to 100 microM cis-, oxali-, or carboplatin. Caspase-3 activity after platinum complex exposure and uptake of the cation ASP+ (4-(4-(diethylamino)styryl)-N-methylpyridinium) of cells on filter membranes and impermeable supports (e.g. culture flasks) were compared. Atomic Force Microscopy (AFM) was used to depict morphometric differences between both culture conditions. RESULTS: In cells on filter membranes, cis-, oxali- and carboplatin induced loss of epithelial monolayer integrity by apoptosis via activation of caspase-3 to different extents. Basolateral application of platinum complexes enhanced toxicity dramatically and uptake of ASP+ from the basolateral side was higher than from the apical medium compartment. Intracellular accumulation of ASP+ was less distinct in cells grown on impermeable supports. Only cisplatin, the most lipophilic investigated complex, induced activation of caspase-3 in these cells. AFM disclosed more prominent cell-cell contacts in cells grown on filter membranes. CONCLUSION: We conclude that toxicity of hydrophilic substances can be underestimated in cells grown on solid supports, if basolateral transport mechanisms are involved. We suggest that unhindered access to basolateral transporters is responsible for higher levels of organic cation uptake and apoptosis in cells on filter membranes, even though more prominent cell-cell contacts indicate a better barrier function.

Animals↗

PubMed2003

Imprinted silencing of Slc22a2 and Slc22a3 does not need transcriptional overlap between Igf2r and Air.

Silencing of the paternal allele of three imprinted genes (Igf2r, Slc22a2 and Slc22a3) requires cis expression of the Air RNA that overlaps the promoter of one of them (Igf2r). Air is a non-coding RNA whose mode of action is unknown. We tested the role of the Igf2r promoter and the role of transcriptional overlap between Igf2r and Air in silencing in this cluster. We analyzed imprinted expression in mice in which the Igf2r promoter is replaced by a thymidine kinase promoter that preserves a transcription overlap with Air, and in mice with a deleted Igf2r promoter that lack any transcriptional overlap with Air. Imprinted silencing of Air, Slc22a2 and Slc22a3 is maintained by the replacement promoter and also in the absence of transcriptional overlap with Air. These results exclude a role for the Igf2r promoter and for transcriptional overlap between Igf2r and Air in silencing Air, Slc22a2 and Slc22a3. Although these results do not completely exclude a role for a double-stranded RNA silencing mechanism, they do allow the possibility that the Air RNA has intrinsic cis silencing properties.

Alleles↗

PubMed2003

Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor, results in early embryonic lethality and chromosomal instability.

The BRCA1 tumor suppressor has been implicated in many cellular pathways, but the mechanisms by which it suppresses tumor formation are not fully understood. In vivo BRCA1 forms a heterodimeric complex with the related BARD1 protein, and its enzymatic activity as a ubiquitin ligase is largely dependent upon its interaction with BARD1. To explore the genetic relationship between BRCA1 and BARD1, we have examined the phenotype of Bard1-null mice. These mice become developmentally retarded and die between embryonic day 7.5 (E7.5) and E8.5. Embryonic lethality results from a severe impairment of cell proliferation that is not accompanied by increased apoptosis. In the absence of p53, the developmental defects associated with Bard1 deficiency are partly ameliorated, and the lethality of Bard1; p53-nullizygous mice is delayed until E9.5. This result, together with the increased chromosomal aneuploidy of Bard1 mutant cells, indicates a role for Bard1 in maintaining genomic stability. The striking similarities between the phenotypes of Bard1-null, Brca1-null, and double Bard1; Brca1-null mice provide strong genetic evidence that the developmental functions of Brca1 and Bard1 are mediated by the Brca1/Bard1 heterodimer.

Aneuploidy↗

PubMed2003

Potential role for ADAM15 in pathological neovascularization in mice.

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.

ADAM Proteins↗

PubMed2002

Placental overgrowth in mice lacking the imprinted gene Ipl.

The Ipl (Tssc3) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the Igf2 gene. Expression of Ipl is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele. Ipl encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (Ipl(neo) and Ipl(loxP)) and another line deleted for the similar but nonimprinted gene Tih1. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the Ipl(neo) allele and was decreased slightly in nulls with the Ipl(loxP) allele. When bred into an Igf2 mutant background, the Ipl deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of Tih1. These results show a nonredundant function for Ipl in restraining placental growth. The data further indicate that Ipl can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size.

Animals↗

PubMed2002

The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity.

Although the protein product of the BRCA1 tumor suppressor gene has been implicated in a surprisingly diverse array of biological processes, the molecular mechanism by which BRCA1 loss promotes tumor formation remains unclear. Nonetheless, a pivotal advance has been achieved by recent studies that establish BRCA1 and its partner polypeptide BARD1 as enzymatic mediators of protein ubiquitination. The potent ubiquitin E3 ligase activity of the BRCA1/BARD1 heterodimer may be responsible for many of the biological properties attributed to BRCA1, including its ability to suppress tumor formation in normal cells.

Amino Acid Sequence↗

PubMed2002

4-[18F]fluoroarylalkylethers via an improved synthesis of n.c.a. 4-[18F]fluorophenol.

This paper describes the improved synthesis of n.c.a. 4- 18F]fluorophenol for the preparation of 18F-labeled alkylarylethers. Nucleophilic fluorination of substituted benzophenone derivatives yielded n.c.a. 4- 18F]fluoro-4'-substituted benzophenones with 80- 90% RCY, which were converted to benzoic acid phenylesters by treatment with peracetic acid. Strong electron-withdrawing substituents like nitro, cyano and trifluoromethyl favor a fluorophenyl-to-oxygen migration resulting in the formation of corresponding benzoic acid fluorophenylesters. N.c.a. 18F]fluorophenol is almost quantitatively formed after hydrolysis and can easily be converted with alkylhalides into n.c.a. 18F]fluoroarylalkylethers.

Alkanes↗

PubMed2002

Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life.

MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.

ADAM Proteins↗