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Using a system optimized for propagating human keratinocytes, culture of skin samples from white and green sturgeons generated epithelial cells capable of making cross-linked protein envelopes. Two distinct forms of TGM1-like mRNA were molecularly cloned from the cells of white sturgeon and detected in green sturgeon cells, accounting for their cellular envelope forming ability. The protein translated from each displayed a cluster of cysteine residues resembling the membrane anchorage region expressed in epidermal cells of teleosts and tetrapods. One of the two mRNA forms (called A) was present at considerably higher levels than the other (called B) in both species. Continuous lines of white sturgeon epidermal cells were established and characterized. Size measurements indicated that a substantial fraction of the cells became enlarged, appearing similar to squames in human epidermal keratinocyte cultures. The cultures also expressed CYP1A, a cytochrome P450 enzyme inducible by activation of aryl hydrocarbon receptor 2 in fish. The cells gradually improved in growth rate over a dozen passages while retaining envelope forming ability, TGM1 expression and CYP1A inducibility. These cell lines are thus potential models for studying evolution of fish epidermis leading to terrestrial adaptation and for testing sturgeon sensitivity to environmental stresses such as pollution.

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

Air pollution poses a serious risk to human health. To help understand the contribution of smoke from wood burning to the harmfulness of air pollution toward the skin, we studied the effects of liquid smoke, aqueous extracts of wood smoke condensate, a commercially available food flavor additive, in cultured keratinocytes. We report that liquid smoke can react with and cross-link keratinocyte cellular proteins, leading to abnormal cross-linked envelope formation. Instead of inducing genes ordinarily involved in terminal differentiation, liquid smoke induced expression of genes associated with stress responses. When transglutaminase activity was inhibited, liquid smoke still promoted protein cross-linking and envelope formation in keratinocytes. This phenomenon likely results from oxidative stress and protein adducts from aldehydes as either preloading the cells with N-acetylcysteine or reducing the aldehyde content of liquid smoke decreased its ability to promote protein cross-linking and envelope formation. Finally, liquid smoke-induced envelopes were found to have elevated protein content, suggesting oxidative cross-linking and formation of protein adducts might impair barrier function by inducing abnormal incorporation of cellular proteins into envelopes. Since the cross-linked protein envelope provides structural stability to the stratum corneum and serves as a scaffold for the organization of the corneocyte lipid envelope (hydrophobic barrier to the environment), these findings provide new insight into the mechanism by which pro-oxidative air pollutants can impair epidermal function.

Polyamines are highly attractive vectors for tumor targeting, particularly with regards to the development of radiolabeled probes for imaging by positron emission (PET) and single-photon emission computed tomography (SPECT). However, the synthesis of selectively functionalized derivatives remains challenging owing to the presence of multiple amino groups of similar re-activity. In this work, we established a synthetic methodology for the selective mono-fluorobenz(o)ylation of various biogenic diamines and polyamines as lead compounds for the perspective development of substrate-based radiotracers for targeting polyamine-specific membrane transporters and enzymes such as transglutaminases. To this end, the polyamine scaffold was constructed by solid-phase synthesis of the corresponding oxopolyamines and subsequent re-duction with BH₃/THF. Primary and secondary amino groups were selectively protected using Dde and Boc as protecting groups, respectively, in orientation to previously reported proce-dures, which enabled the selective introduction of the reporter groups. For example, N¹-FBz-spermidine, N⁴-FBz-spermidine, N⁸-FBz-spermidine, and N¹-FBz-spermine and N⁴-FBz-spermine (FBz=4-fluorobenzoyl) were obtained in good yields by this approach. The advantages and disadvantages of this synthetic approach are discussed in detail and its suitability for radiolabeling was demonstrated for the solid-phase synthesis of N¹-[¹⁸F]FBz-cadaverine.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Defects in keratinocyte transglutaminase (TGM1), resulting in an improper protein scaffold for deposition of the lipid barrier, comprise a major source of autosomal recessive congenital ichthyosis. For that reason, the composition and formation of the cornified (cross-linked) protein envelope of the epidermis have been of considerable interest. Since the isopeptide cross-linked protein components are not individually isolable once incorporated, purified envelopes were analysed by mass spectrometry after trypsin digestion. Quantitative estimates of the identified components revealed some 170 proteins, each comprising at least 0.001% of the total, of which keratins were major constituents accounting for ≈74% of the total. Some prevalent non-keratin constituents such as keratinocyte proline-rich protein, loricrin and late envelope protein-7 were preferentially incorporated into envelopes. The results suggest a model where, as previously observed in hair shaft and nail plate, a diversity of cellular proteins are incorporated. They also help rationalize the minimal effect on epidermis of ablating genes for specific single envelope structural components. The quantitative profile of constituent proteins provides a foundation for future exploration of envelope perturbations that may occur in pathological conditions.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Defects in keratinocyte transglutaminase (TGM1), resulting in an improper protein scaffold for deposition of the lipid barrier, comprise a major source of autosomal recessive congenital ichthyosis. For that reason, the composition and formation of the cornified (cross-linked) protein envelope of the epidermis have been of considerable interest. Since the isopeptide cross-linked protein components are not individually isolable once incorporated, purified envelopes were analysed by mass spectrometry after trypsin digestion. Quantitative estimates of the identified components revealed some 170 proteins, each comprising at least 0.001% of the total, of which keratins were major constituents accounting for ≈74% of the total. Some prevalent non-keratin constituents such as keratinocyte proline-rich protein, loricrin and late envelope protein-7 were preferentially incorporated into envelopes. The results suggest a model where, as previously observed in hair shaft and nail plate, a diversity of cellular proteins are incorporated. They also help rationalize the minimal effect on epidermis of ablating genes for specific single envelope structural components. The quantitative profile of constituent proteins provides a foundation for future exploration of envelope perturbations that may occur in pathological conditions.

Numerous genetic conditions give rise to a scaly skin phenotype as a result of impaired barrier function. Present work investigates the degree to which the departure from normal of ichthyosis corneocytes on the skin surface depends upon the basic defect as judged by proteomic profiling. Analyzing autosomal recessive congenital ichthyosis arising from defects in the genes PNPLA1, SDR9C7 and TGM1 revealed that profiles of PNPLA1 samples displayed the greatest degree of departure from normal control epidermis, with SDR9C7 samples nearly as divergent, and TGM1 the least divergent. Although the profiles were distinctive, each displaying a set of altered protein levels, they exhibited alterations in 20 proteins in common, of which 15 were expressed consistently at higher and 5 at lower levels. Departure from the normal profile was examined at three different anatomic sites (forearm, forehead, leg). Reflecting that the normal protein profile differed at these sites, comparing profiles from afflicted subjects revealed that the degree of alteration in profile was site-dependent. These results suggest proteomic profiling can provide a quantitative measure of departure from the normal state of epidermis. Further development may help characterize consequences of the genetic defects, including perturbation of signaling pathways, and supplement visual evaluation of treatment. SIGNIFICANCE: ARCI are rare cornification disorders caused by mutations in at least 14 different genes leading to perturbed metabolism and organization of constituent biomolecules of cornified envelopes. The phenotypic manifestations of the disorder vary among individuals with the same as well as different genetic defects and even at different anatomic sites within the same individual. The present study investigates the proteomic disturbances at three anatomic sites in patients carrying mutations in three different genes. Our findings provide a basis for elucidating genotype to proteome relationships for ARC

Advances in mass spectrometry-based proteomics now permit analysis of complex cellular structures. Application to epidermis and its appendages (nail plate, hair shaft) has revealed a wealth of information about their protein profiles. The results confirm known site-specific differences in levels of certain keratins and add great depth to our knowledge of site specificity of scores of other proteins, thereby connecting anatomy and pathology. An example is the evident overlap in protein profiles of hair shaft and nail plate, helping rationalize their sharing of certain dystrophic syndromes distinct from epidermis. In addition, interindividual differences in protein level are manifest as would be expected. This approach permits characterization of altered profiles as a result of disease, where the magnitude of perturbation can be quantified and monitored during treatment. Proteomic analysis has also clarified the nature of the isopeptide cross-linked residual insoluble material after vigorous extraction with protein denaturants, nearly intractable to analysis without fragmentation. These structures, including the cross-linked envelope of epidermal corneocytes, are comprised of hundreds of protein constituents, evidence for strengthening the terminal structure complementary to disulphide bonding. Along with other developing technologies, proteomic analysis is anticipated to find use in disease risk stratification, detection, diagnosis and prognosis after the discovery phase and clinical validation.

Among the adaptations of aquatic species during evolution of terrestrial tetrapods was the development of an epidermis preventing desiccation. In present day mammals, keratinocytes of the epidermis, using a membrane-bound transglutaminase (Tgm1), accomplish this function by synthesizing a scaffold of cross-linked protein to which a lipid envelope is attached. This study characterizes the abilities of two homologous transglutaminase isozymes in the teleost fish tilapia to form cross-linked protein structures and their expression in certain tissues. Results indicate they are capable of membrane localization and of generating cellular structures resistant to detergent solubilization. They are both expressed in epithelial cells of the lip, buccal cavity and tips of gill filaments. Adaptation of transglutaminase use in evolution of terrestrial keratinocytes evidently involved refinements in tissue expression, access to suitable substrate proteins and activation of cross-linking during terminal differentiation.

Among the adaptations of aquatic species during evolution of terrestrial tetrapods was the development of an epidermis preventing desiccation. In present day mammals, keratinocytes of the epidermis, using a membrane-bound transglutaminase (Tgm1), accomplish this function by synthesizing a scaffold of cross-linked protein to which a lipid envelope is attached. This study characterizes the abilities of two homologous transglutaminase isozymes in the teleost fish tilapia to form cross-linked protein structures and their expression in certain tissues. Results indicate they are capable of membrane localization and of generating cellular structures resistant to detergent solubilization. They are both expressed in epithelial cells of the lip, buccal cavity and tips of gill filaments. Adaptation of transglutaminase use in evolution of terrestrial keratinocytes evidently involved refinements in tissue expression, access to suitable substrate proteins and activation of cross-linking during terminal differentiation.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy‐based approach for the determination of TGase 2’s transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein‐ and rhodamine B‐conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid‐phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L‐lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z’ factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy‐based approach for the determination of TGase 2’s transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein‐ and rhodamine B‐conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid‐phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L‐lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z’ factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Importance: Bathing suit ichthyosis (BSI) is a rare congenital disorder of keratinization characterized by restriction of scale to sites of relatively higher temperature such as the trunk, with cooler areas remaining unaffected. Fewer than 40 cases have been reported in the literature. Bathing suit ichthyosis is caused by recessive, temperature-sensitive mutations in the transglutaminase-1 gene (TGM1). Clear genotype-phenotype correlations have been difficult to establish because several of the same TGM1 mutations have been reported in BSI and other forms of congenital ichthyosis. We identify novel and recurrent mutations in 16 participants with BSI. Objective: To expand the genotypic spectrum of BSI, identifying novel TGM1 mutations in patients with BSI, and to use BSI genotypes to draw inferences about the temperature sensitivity of TGM1 mutations. Design, Setting, and Participants: A total of 16 participants with BSI from 13 kindreds were identified from 6 academic medical centers. A detailed clinical history was obtained from each participant, including phenotypic presentation at birth and disease course. Each participant underwent targeted sequencing of TGM1. Main Outcomes and Measures: Phenotypic and genotypic characteristics in these patients from birth onward. Results: Of the 16 participants, 7 were male, and 9 were female (mean age, 12.6 years; range, 1-39 years). We found 1 novel TGM1 indel mutation (Ile469_Cys471delinsMetLeu) and 8 TGM1 missense mutations that to our knowledge have not been previously reported in BSI: 5 have been previously described in non-temperature-sensitive forms of congenital ichthyosis (Arg143Cys, Gly218Ser, Gly278Arg, Arg286Gln, and Ser358Arg), and 3 (Tyr374Cys, Phe495Leu, and Ser772Arg) are novel mutations. Three probands were homozygous for Arg264Trp, Arg286Gln, or Arg315Leu, indicating that these mutations are temperature sensitive. Seven of 10 probands with a compound heterozygous TGM1 genotype had a mutation at either arginin

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

Patients with psoriasis have been shown to have a higher prevalence of other autoimmune diseases including celiac disease, a condition marked by sensitivity to dietary gluten. A number of studies suggest that psoriasis and celiac disease share common genetic and inflammatory pathways. Here we review the epidemiologic association between psoriasis and celiac disease and perform a meta-analysis to determine whether patients with psoriasis more frequently harbor serologic markers of celiac disease. We also examine whether a gluten-free diet can improve psoriatic skin disease.

OBJECTIVES: Undernourishment is common in children with cerebral palsy (CP), but the reasons are unknown. We previously reported elevated levels of immunoglobulin (Ig) A and IgG antibodies against gliadin (AGA) and tissue transglutaminase (tTG) in 99 children and young adults with CP without characteristic findings of gluten enteropathy in small bowel biopsies. Our aim was to perform a case-control study of IgG antibodies against other dietary antigens, AGA, anti-tTG, and IgE antibodies against wheat and gluten. METHODS: Sera from 99 cases with CP and 99 healthy, age- and sex-matched controls were analysed with fluorescence enzyme-linked immunosorbent assay for detection of IgG antibodies against β-lactoglobulin, casein, egg white, IgG- and IgA-AGA, IgA-anti-tTG, and IgE antibodies against gluten and wheat. RESULTS: Compared with controls, the odds ratio in cases with CP for having elevated levels of IgG antibodies against β-lactoglobulin was 17.0 (95% confidence interval [CI] 2.3-128), against casein 11.0 (95% CI 2.6-46.8), and against egg white 7.0 (95% CI 1.6-30.8). The IgE responses for wheat/gluten were generally low. The tetraplegic and dyskinetic CP subtypes had significantly higher frequencies of elevated levels for all of the tested antibodies except IgG against egg white, and IgA-anti-tTG. A significantly lower weight was seen in cases with CP with positive versus negative serology. CONCLUSIONS: Elevated levels of IgG against dietary antigens were more frequent in the CP group compared with controls, and particularly in the tetraplegic and dyskinetic CP subtypes with the most severe neurologic handicap and undernourishment. Hypothetically, malnourishment may cause increased intestinal permeability and thus immunization against dietary antigens., Funding agencies are:Research Committee of Orebro County Council Folke Bernadotte Foundation, Stockholm, Sweden

To explore the usefulness of protein profiling for characterization of ichthyoses, we here determined the profile of human epidermal stratum corneum by shotgun proteomics. Samples were analyzed after collection on tape circles from six anatomic sites (forearm, palm, lower leg, forehead, abdomen, upper back), demonstrating site-specific differences in profiles. Additional samples were collected from the forearms of subjects with ichthyosis vulgaris (filaggrin (FLG) deficiency), recessive X-linked ichthyosis (steroid sulfatase (STS) deficiency) and autosomal recessive congenital ichthyosis type lamellar ichthyosis (transglutaminase 1 (TGM1) deficiency). The ichthyosis protein expression patterns were readily distinguishable from each other and from phenotypically normal epidermis. In general, the degree of departure from normal was lower from ichthyosis vulgaris than from lamellar ichthyosis, parallel to the severity of the phenotype. Analysis of samples from families with ichthyosis vulgaris and concomitant modifying gene mutations (STS deficiency, GJB2 deficiency) permitted correlation of alterations in protein profile with more complex genetic constellations.

This PhD thesis presents novel and original research in the field of Insulin-like Growth Factor-I (or IGF-I) biology. IGF-I plays an essential role in promoting normal human growth and development; it also represents both a target and treatment for various diseases. This thesis provides interesting insights into previously uncharacterised mechanisms of action that underlie IGF-I biology. Such findings may lead to improved and novel treatments across a broad range of medical conditions.

Bronchopulmonary dysplasia is a complication of premature birth characterized by impaired alveolar development. Remodeling of the ECM is a driving force for alveolarization and, if pertubated, may impair septation, suggesting dysregulation of ECM remodeling enzymes that drive collagen fiber formation and maturation: the procollagen-lysine, 2-oxoglutarate 5-dioxygenases (Plod) family, also known as lysyl hydroxylases (which catalyzes glycosylation and hydroxylation of collagen), and the transglutaminases Tgm1 and Tgm2, which cross-link ECM components. Expressions of Plod1, Plod2, Plod3, Tgm1 and Tgm2 were determined using a popular mouse model of BPD, in which mouse pups are exposed to hyperoxia (85% O2) or normoxia (21% O2) for 28 days after birth. The lungs of these mice were harvested at various time-points and assessed for Plod and Tgm expression and localization by semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. Increased expressions of Plods and Tgms could be observed at the gene and protein levels under hyperoxic conditions compared to normoxic conditions. The data suggest that BPD is characterized by elevated levels of ECM-stabilizing molecules, which may make the ECM more resistant to remodeling. This overstabilized state of the ECM may, at least in part, underlie the arrested septation observed both in the lungs of infants with BPD, and in animal models of BPD., Bronchopulmonale Dysplasie ist eine Komplikation bei Frühgeborenen, welche durch eine fehlgesteuerte Entwicklung der Lungenalveolen charakterisiert ist. Der Umbau der ECM ist die treibende Kraft für die Bildung der Alveolen und kann, falls er behindert wird, zu einer Beeinträchtigung der alveolären Septierung führen. Dies lässt auf eine Fehlregulation der für den Umbau der ECM verantwortlichen Enzyme bei der Entstehung der BPD schließen: Die Prokollagen-Lysin, 2-Oxoglutarat 5-Dioxygenase (Plod) Familie, auch bekannt als Lysyl Hydroxylasen (welche die Glykosylierung und Hydroxylierung von Kollagenmolekülen katalysieren) und die Transglutaminasen Tgm1 und Tgm2, welche die ECM Komponenten quervernetzen. Um die Expression von Plod1, Plod2, Plod3, Tgm1 und Tgm2 zu untersuchen, wurde ein gängiges Maus-Modell der BPD benutzt, bei dem neugeborene Mäuse in den ersten 28 postnatalen Tagen entweder hohen Sauerstoff-Konzentrationen (85% O2) oder normalen Sauerstoff-Konzentrationen (21% O2) ausgesetzt wurden. Die Lungen dieser Mäuse wurden zu verschiedenen Zeitpunkten entnommen und mittels semiquantitativer RT-PCR, Western-Blot und Immunhistochemie hinsichtlich der Plod- und Tgm-Expression und -Lokalisation untersucht. Unter dem Einfluss von hohen Sauerstoffkonzentrationen konnte eine Steigerung der Plod- und Tgm-Expression sowohl auf Gen- als auch auf Proteinebene beobachtet werden, verglichen mit der Expression in der Kontrolle unter normalen Sauerstoffkonzentrationen. Diese Daten deuten darauf hin, dass BPD durch eine gesteigerte Synthese von ECM-stabilisierenden Enzymen gekennzeichnet ist, was möglicherweise zu einer Behinderung des notwendigen Umbaus der ECM führt. Dieser „über-stabilisierte“ Zustand könnte, zumindest teilweise, der frühzeitigen Terminierung der alveolären Septierung zu Grunde liegen, welche sowohl in den Lungen der an BPD verstorbenen Kinder, als auch in BPD Tiermodellen beobachtet wurde.

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

Background: Changing perspectives on the natural history of celiac disease (CD), new serology and genetic tests, and amended histological criteria for diagnosis cast doubt on past prevalence estimates for CD. We set out to establish a more accurate prevalence estimate for CD using a novel serogenetic approach.Methods: The human leukocyte antigen (HLA)-DQ genotype was determined in 356 patients with 'biopsy-confirmed' CD, and in two age-stratified, randomly selected community cohorts of 1,390 women and 1,158 men. Sera were screened for CD-specific serology.Results: Only five 'biopsy-confirmed' patients with CD did not possess the susceptibility alleles HLA-DQ2.5, DQ8, or DQ2.2, and four of these were misdiagnoses. HLA-DQ2.5, DQ8, or DQ2.2 was present in 56% of all women and men in the community cohorts. Transglutaminase (TG)-2 IgA and composite TG2/deamidated gliadin peptide (DGP) IgA/IgG were abnormal in 4.6% and 5.6%, respectively, of the community women and 6.9% and 6.9%, respectively, of the community men, but in the screen-positive group, only 71% and 75%, respectively, of women and 65% and 63%, respectively, of men possessed HLA-DQ2.5, DQ8, or DQ2.2. Medical review was possible for 41% of seropositive women and 50% of seropositive men, and led to biopsy-confirmed CD in 10 women (0.7%) and 6 men (0.5%), but based on relative risk for HLA-DQ2.5, DQ8, or DQ2.2 in all TG2 IgA or TG2/DGP IgA/IgG screen-positive subjects, CD affected 1.3% or 1.9%, respectively, of females and 1.3% or 1.2%, respectively, of men. Serogenetic data from these community cohorts indicated that testing screen positives for HLA-DQ, or carrying out HLA-DQ and further serology, could have reduced unnecessary gastroscopies due to false-positive serology by at least 40% and by over 70%, respectively.Conclusions: Screening with TG2 IgA serology and requiring biopsy confirmation caused the community prevalence of CD to be substantially underestimated. Testing for HLA-DQ genes and confirmatory sero

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.

To explore the usefulness of protein profiling for characterization of ichthyoses, we here determined the profile of human epidermal stratum corneum by shotgun proteomics. Samples were analyzed after collection on tape circles from six anatomic sites (forearm, palm, lower leg, forehead, abdomen, upper back), demonstrating site-specific differences in profiles. Additional samples were collected from the forearms of subjects with ichthyosis vulgaris (filaggrin (FLG) deficiency), recessive X-linked ichthyosis (steroid sulfatase (STS) deficiency) and autosomal recessive congenital ichthyosis type lamellar ichthyosis (transglutaminase 1 (TGM1) deficiency). The ichthyosis protein expression patterns were readily distinguishable from each other and from phenotypically normal epidermis. In general, the degree of departure from normal was lower from ichthyosis vulgaris than from lamellar ichthyosis, parallel to the severity of the phenotype. Analysis of samples from families with ichthyosis vulgaris and concomitant modifying gene mutations (STS deficiency, GJB2 deficiency) permitted correlation of alterations in protein profile with more complex genetic constellations.

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by whic

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by whic

OBJECTIVES: The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro . METHODS: Human primary trophoblastic cells, isolated from term placenta, were exposed to anti-tTG immunoglobulin G (IgG) antibodies, both commercially available and separated from sera of three untreated celiac women. The ability of anti-tTG antibodies to bind to trophoblastic cells, invasiveness of placental cells through a layer of extracellular matrix, and the activity of cellular matrix metalloprotease (MMP) and cellular apoptosis were evaluated, as indicators of trophoblast damage, by TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) and annexin V expression. RESULTS: Anti-tTG IgG showed a specifi c dose- and time-dependent binding to human trophoblast. In addition, trophoblastic cells, after being exposed to anti-tTG IgG antibodies, both commercially available and separated from sera of celiac women, showed an impaired invasiveness, a decreased activity of cellular MMP, and a greater percentage of TUNEL positivity and annexin V positivity. CONCLUSIONS: We showed that the binding of anti-tTG antibodies to trophoblast might represent a key mechanism by which the embryo implantation and pregnancy outcome are impaired in untreated celiac pregnant women. Because healthy trophoblast development is essential for placental and fetal development, these data provide a novel mechanism for CD-induced infertility, early pregnancy loss, and intrauterine growth retardation.

OBJECTIVES: The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro. METHODS: Human primary trophoblastic cells, isolated from term placenta, were exposed to anti-tTG immunoglobulin G (IgG) antibodies, both commercially available and separated from sera of three untreated celiac women. The ability of anti-tTG antibodies to bind to trophoblastic cells, invasiveness of placental cells through a layer of extracellular matrix, and the activity of cellular matrix metalloprotease (MMP) and cellular apoptosis were evaluated, as indicators of trophoblast damage, by TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) and annexin V expression. RESULTS: Anti-tTG IgG showed a specific dose- and time-dependent binding to human trophoblast. In addition, trophoblastic cells, after being exposed to anti-tTG IgG antibodies, both commercially available and separated from sera of celiac women, showed an impaired invasiveness, a decreased activity of cellular MMP, and a greater percentage of TUNEL positivity and annexin V positivity. CONCLUSIONS: We showed that the binding of anti-tTG antibodies to trophoblast might represent a key mechanism by which the embryo implantation and pregnancy outcome are impaired in untreated celiac pregnant women. Because healthy trophoblast development is essential for placental and fetal development, these data provide a novel mechanism for CD-induced infertility, early pregnancy loss, and intrauterine growth retardation.

The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro.

In order to elucidate the molecular mechanism of transglutaminase-mediated myosin cross-linking, a fluorescent monodansylcadaverine (MDC) was incorporated into carp Cyprinus carpio myosin and the reactive Gln residues were analyzed by cyanogen bromide cleavage. The fluorescence was predominantly detected in a 10.5 kDa BrCN-fragment, which is assumed to be located in subfragment 2 of the myosin heavy chain. Furthermore, lysyl endopeptidase digestion of the 10.5 kDa fragment revealed that MDC was specifically incorporated into the 520th Gln residue of the subfragment 2 domain. When meat paste prepared from walleye pollack Theragra chalcogramma frozen surimi was incubated with MDC, the fluorescence was mostly observed in a 16 kDa BrCN-fragment and also slightly detected in other three bands. By the digestion of 16 kDa fragment with lysyl endopeptidase, it was elucidated that MDC was incorporated specifically into Gln-520 of myosin subfragment 2, as well as detected in carp. This domain around Gln-520 is likely to be a common critical region for dimer formation of myosin heavy chains for both fish species. In walleye pollack, other reactive Gln residues are presumed to be exist in the C-terminus of the light meromyosin. This slight difference may be significant in a capacity to form tetramers or even larger multimers.

In order to elucidate the molecular mechanism of transglutaminase-mediated myosin cross-linking, a fluorescent monodansylcadaverine (MDC) was incorporated into carp Cyprinus carpio myosin and the reactive Gln residues were analyzed by cyanogen bromide cleavage. The fluorescence was predominantly detected in a 10.5 kDa BrCN-fragment, which is assumed to be located in subfragment 2 of the myosin heavy chain. Furthermore, lysyl endopeptidase digestion of the 10.5 kDa fragment revealed that MDC was specifically incorporated into the 520th Gln residue of the subfragment 2 domain. When meat paste prepared from walleye pollack Theragra chalcogramma frozen surimi was incubated with MDC, the fluorescence was mostly observed in a 16 kDa BrCN-fragment and also slightly detected in other three bands. By the digestion of 16 kDa fragment with lysyl endopeptidase, it was elucidated that MDC was incorporated specifically into Gln-520 of myosin subfragment 2, as well as detected in carp. This domain around Gln-520 is likely to be a common critical region for dimer formation of myosin heavy chains for both fish species. In walleye pollack, other reactive Gln residues are presumed to be exist in the C-terminus of the light meromyosin. This slight difference may be significant in a capacity to form tetramers or even larger multimers.

BackgroundTGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment.MethodsIn vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes.ResultsIn vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity.ConclusionsA

BackgroundTGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment.MethodsIn vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes.ResultsIn vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity.ConclusionsA

Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.

All novel proteins must be assessed for their potential allergenicity before they are introduced into the food market. One method to achieve this is the 2001 FAO/WHO Decision Tree recommended for evaluation of proteins from genetically modified organisms (GMOs). It was the aim of this study to investigate the allergenicity of microbial transglutaminase (m-TG) from Streptoverticillium mobaraense. Amino acid sequence similarity to known allergens, pepsin resistance, and detection of protein binding to specific serum immunoglobulin E (IgE) (RAST) have been evaluated as recommended by the decision tree. Allergenicity in the source material was thought unlikely, since no IgE-mediated allergy to any bacteria has been reported. m-TG is fully degraded after 5 min of pepsin treatment. A database search showed that the enzyme has no homology with known allergens, down to a match of six contiguous amino acids, which meets the requirements of the decision tree. However, there is a match at the five contiguous amino acid level to the major codfish allergen Gad c1. The potential cross reactivity between m-TG and Gad c1 was investigated in RAST using sera from 25 documented cod-allergic patients and an extract of raw codfish. No binding between patient IgE and m-TG was observed. It can be concluded that no safety concerns with regard to the allergenic potential of m-TG were identified.

All novel proteins must be assessed for their potential allergenicity before they are introduced into the food market. One method to achieve this is the 2001 FAO/WHO Decision Tree recommended for evaluation of proteins from genetically modified organisms (GMOs). It was the aim of this study to investigate the allergenicity of microbial transglutaminase (m-TG) from Streptoverticillium mobaraense. Amino acid sequence similarity to known allergens, pepsin resistance, and detection of protein binding to specific serum immunoglobulin E (IgE) (RAST) have been evaluated as recommended by the decision tree. Allergenicity in the source material was thought unlikely, since no IgE-mediated allergy to any bacteria has been reported. m-TG is fully degraded after 5 min of pepsin treatment. A database search showed that the enzyme has no homology with known allergens, down to a match of six contiguous amino acids, which meets the requirements of the decision tree. However, there is a match at the five contiguous amino acid level to the major codfish allergen Gad c1. The potential cross reactivity between m-TG and Gad c1 was investigated in RAST using sera from 25 documented cod-allergic patients and an extract of raw codfish. No binding between patient IgE and m-TG was observed. It can be concluded that no safety concerns with regard to the allergenic potential of m-TG were identified.

In this study transglutaminase was used to achieve ε-γ-(glutamyl) lysine cross-linking of milk proteins, in Trim™ and Full Fat milks, the same milks with a variety of added protein concentrates, and finally in yogurt and Petite Suisse acid gel systems. The effects of a preheat treatment, enzyme incubation temperature, enzyme inactivation after the enzyme incubation period and homogenization on the cross-linking of the three major casein and two whey proteins were also studied. The degree of cross-linking was established by the use of SDS PAGE gel electrophoresis. The results indicated that cross-linking of the major casein and whey proteins was maximized if the milk was preheated for 10 minutes at 90°C and then cooled before addition of the transglutaminase. However, the preheat treatment was not always advantageous in Trim™ milk systems, but was essential for Full Fat milk systems. Maximal cross-linking of milk proteins occurred if the enzyme/milk system was incubated at 37°C for two hours rather than at 55°C for the same period. The extent of cross-linking increased in an almost linear fashion with increasing transglutainase concentration in most milk systems, with maximal cross-linking occurring when the enzyme concentration was 100 U/mL. Studies on one milk system showed that whey loss and gel strength deteriorated if more than 100 U/mL of enzyme was used. The study demonstrated that homogenization was an essential step for protein cross-linking if the system contained any fat. Casein and whey protein transglutaminase mediated cross-linking was maximized in Full Fat milk systems if the milk was homogenized before transglutaminase was added. Maximal cross-linking, particularly of whey proteins, occurred in Full Fat milk systems if the milk was preheated for 10 minutes at 90°C, cooled to 60°C and then homogenized at 50/150, cooled further to 37°C and then incubated with 100 U/mL of enzyme for two hours. Addition of sodium caseinate or milk protein isolate to Trim™

The ky mouse mutant exhibits a primary degenerative myopathy preceding chronic thoraco-lumbar kyphoscoliosis. The histopathology of the ky mutant suggests that Ky protein activity is crucial for normal muscle growth and function as well as the maturation and stabilization of the neuromuscular junction. Muscle hypertrophy in response to increasing demand is deficient in the ky mutant, whereas adaptive fibre type shifts take place. The ky locus has previously been localized to a small region of mouse chromosome 9 and we have now identified the gene and the mutation underlying the kyphoscoliotic mouse. The ky transcript encodes a novel protein that is detected only in skeletal muscle and heart. The identification of the ky gene will allow detailed analysis of the impact of primary myopathy on idiopathic scoliosis in mice and man.

Angiogenesis is vital to tumor growth and metastasis. The scope of this grant was to study in detail the role of Tissue transglutaminase (tTG) during wound healing and tumorigenesis. In the first year of the grant proposal, we have described the expression, localization, molecular form and tTG's association with other major determinants of wound healing and tumorigenesis. Our findings clearly show that tTG is readily upregulated in wound healing and rat mammary adenocarcinoma and is associated with endothelial and inflammatory cells. Hypoxia, Vascular endothelial growth factor, Transforming growth factor beta and Tumor necrosis factor alpha are also upregulated alongside tTG in those cells. tTG is quickly proteolysed in the tissues and that may have important consequences as tTC can hydrolyze ATP/GTP in its fragmented form. In the second year and continuing, We have also shown that tTG can inhibit tumor growth as part of host response mechanism and identified that tTG's effects are compartmentalized i.e. host and tumor. In tumor cells, it prevents growth and metastasis at primary site and promotes establishment of metastasis once the tumor cells escape from the primary focus. We are still continuing with establishing the mechanism behind tTG effects.

Enhanced apoptosis characterizes several pathologies affecting human liver. This study sought to determine whether apoptosis is involved in the formation of fibrotic lesions occurring in hepatic disease. The expression of Bcl-2 was analysed, and of 'tissue' transglutaminase (tTG), a cross-linking enzyme which recent evidence suggests plays a role in the formation of fibrotic lesions in experimental settings. Regardless of the degree of liver injury, tTG abnormally accumulated in the liver cells adjacent to fibrotic tissue. Many cells showing DNA fragmentation and morphological features of apoptosis were also observed near fibrotic lesions. Bcl-2 was detected predominantly in infiltrating lymphocytes within the liver tissue. Marked staining for both tTG protein and chromatin was also observed in the acellular fibrotic tissue, which suggested an active release of intracellular macromolecules from the dying cells into the extracellular matrix. This study indicates that fibrogenesis in the liver is associated with the release of tTG from dying cells. By cross-linking extracellular matrix proteins, this enzyme might play a role in the formation of fibrotic lesions.

A 13-year-old boy presented with a polypoid nodule localized in the groin. Although the clinical and histopathological features corresponded to previously described diagnostic criteria of epithelioid cell histiocytoma, immunohistochemical and ultrastructural findings suggested vascular differentiation of the epithelioid cells. In particular, the immunohistochemical negativity of the epithelioid cell elements for Factor XIIIa failed to substantiate the previously forwarded hypothesis that this lesion represents a dendrocytoma. Instead, the presence of histiocytoid, vacuolated cells occasionally containing sparse red blood cells, positive staining for Factor VIII-related antigen, and ultrastructural evidence of endothelial characteristics in epithelioid neoplastic cells favor the hypothesis that "epithelioid cell histiocytoma" is of endothelial origin. We suggest the descriptive term cutaneous histiocytoid hemangioendothelioma for this lesion.

Human keratinocytes express a particulate transglutaminase that can be released from the membrane by limited proteolysis with trypsin or plasmin to yield a form that is congruent to 80 kDa. The enzyme from cultured cells was also releasable by endogenous proteolysis to yield a catalytically active fragment of congruent to 80 kDa. Endogenous release was strongly dependent upon temperature and Ca2+ concentration and was inhibited by iodoacetate, but not by leupeptin, antipain or phenylmethanesulphonyl fluoride. These phenomena raise the possibility of partial translocation of transglutaminase activity to the cytoplasm by proteolysis to which the enzyme is subject during terminal differentiation. In addition, hydrodynamic measurements showed that the endogenously released enzyme was monomeric in solution (79 kDa), whereas that solubilized by hydroxylamine without proteolysis appeared dimeric (190 kDa). The latter dimeric state may reflect either an altered conformation of the enzyme or post-translational modification beyond fatty acid esterification.

Human keratinocytes express a particulate transglutaminase that can be released from the membrane by limited proteolysis with trypsin or plasmin to yield a form that is congruent to 80 kDa. The enzyme from cultured cells was also releasable by endogenous proteolysis to yield a catalytically active fragment of congruent to 80 kDa. Endogenous release was strongly dependent upon temperature and Ca2+ concentration and was inhibited by iodoacetate, but not by leupeptin, antipain or phenylmethanesulphonyl fluoride. These phenomena raise the possibility of partial translocation of transglutaminase activity to the cytoplasm by proteolysis to which the enzyme is subject during terminal differentiation. In addition, hydrodynamic measurements showed that the endogenously released enzyme was monomeric in solution (79 kDa), whereas that solubilized by hydroxylamine without proteolysis appeared dimeric (190 kDa). The latter dimeric state may reflect either an altered conformation of the enzyme or post-translational modification beyond fatty acid esterification.

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: during the differentiation of keratinocytes, during pregnancy, being evident principally in the endometrium but detectable elsewhere as well, and during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.

The predominant form of the cross-linking enzyme, transglutaminase, in cultured normal human epidermal keratinocytes, is found in cell particulate material and can be solubilized by nonionic detergent. It elutes as a single peak upon either anion-exchange or gel-filtration chromatography. Monoclonal antibodies raised to the particulate enzyme cross-react with one of two transglutaminases in the cell cytosol. The second cytosolic transglutaminase, which has distinct kinetic and physical properties from the first, does not cross-react and is not essential for formation of the keratinocyte cross-linked envelope in vitro. The anti-transglutaminase antibodies stain the more differentiated layers of epidermis in a pattern similar to that given by anti-involucrin antiserum. These observations support the hypothesis that the transglutaminase so identified is involved in cross-linked envelope formation in vivo.

Epithelial cells derived from a variety of glandular and other nonkeratinized rat tissues (pituitary, thyroid, bladder, endometrium, trachea, seminal vesicle, prostate, and mammary epithelium) were serially cultivated using a feeder layer of lethally irradiated 3T3 cells. The epithelial cells grew as progressively expanding colonies, in some cases stratified, and were shown to form cornified envelopes upon ionophore-induced activation of cross-linking. Cultures derived from each tissue were distinguishable from the others by characteristic cellular appearance and colony morphology. Those examined in greater detail could be distinguished biochemically in three ways. (a) A majority of cells in sparse cultures of bladder, tracheal, endometrial, and vaginal epithelial cells were capable of envelope formation, whereas those from pituitary, thyroid, seminal vesicle, and mammary epithelia did not attain maximal envelope forming ability until after confluence. (b) Bladder, thyroid, and pituitary cells exhibited different electrophoretic profiles of keratins, which accounted for 20-50% of the cellular protein. (c) Bladder cells were distinguished from thyroid and pituitary cells by a greater suppression of envelope-forming ability by vitamin A. These observations showed that cells from many epithelia have the potential to express properties of keratinocytes in culture while maintaining morphological and physiological differences. Serial passage of these cells generated continuous lines.