Heterozygous frameshift mutation in keratin 5 in a family with Galli-Galli disease.

BACKGROUND: Reticulate pigmentary disorders include the rare autosomal dominant Galli-Galli disease (GGD) and Dowling-Degos disease (DDD). Clinical diagnosis between some of the subtypes can be difficult due to a degree of overlap between clinical features, therefore analysis at the molecular level may be necessary to confirm the diagnosis. OBJECTIVES: To identify the underlying genetic defect in a 48-year-old Asian-American woman with a clinical diagnosis of GGD. METHODS: Histological analysis was performed on a skin biopsy using haematoxylin-eosin staining. KRT5 (the gene encoding keratin 5) was amplified from genomic DNA and directly sequenced. RESULTS: The patient had a history of pruritus and hyperpigmented erythematous macules and thin papules along the flexor surfaces of her arms, her upper back and neck, axillae and inframammary areas. Hypopigmented macules were seen among the hyperpigmentation. A heterozygous 1-bp insertion mutation in KRT5 (c.38dupG; p.Ser14GlnfsTer3) was identified in the proband. This mutation occurs within the head domain of the keratin 5 protein leading to a frameshift and premature stop codon. CONCLUSIONS: From the histological findings and mutation analysis the individual was identified as having GGD due to haploinsufficiency of keratin 5.

Proteasome inhibitors modulate chemokine production in lung epithelial and monocytic cells.

Proteasome inhibition has become a target for antitumour and anti-inflammatory therapy. The present study investigated the influence of cysteine proteinase and proteasome inhibitors on chemokine production in lung epithelial cells and monocytic cells. The lung carcinoma cell lines A549, SK-MES, NCI-H727, virus-transformed bronchial epithelial cell line BEAS-2B, primary lung epithelial cells, and the acute monocytic leukaemia cell lines Mono-Mac-6 and THP-1 were incubated with proteasome (N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN), beta-lactone) or cysteine proteinase inhibitor (L-trans-Epoxysuccinyl-Leu-3-methylbutylamide-ethyl ester) and the influence on chemokine production (interleukin-8: IL-8, monocyte chemoattractant protein-1, RANTES) was quantified at protein and mRNA levels. Inhibition of proteasome activity by ALLN and beta-lactone resulted in significantly increased IL-8 secretion (5- to 22-fold). Cysteine proteinase inhibitors did not influence chemokine production. The simultaneous rise in IL-8 mRNA was caused by an increased half-life of mRNA and increased RNA synthesis. Moreover, analysis of transcription factor activation revealed induction of activator protein-1 (c-Jun) activity by proteasome inhibition, whereas nuclear factor-kappaB (p50 and p65) was not activated. The significant increase in IL-8 production after proteasome inhibition was also observed in primary lung epithelial cells and in monocytic cells. In addition, the secreted IL-8 was biologically active as shown by the neutrophil chemotaxis assay. In conclusion, it was shown that proteasome inhibitors stimulate interleukin-8 secretion in lung epithelial cells and monocytic cells, thus recruiting neutrophils.

Lysosomal cysteine proteases in the lung: role in protein processing and immunoregulation.

Lysosomal cysteine proteases are a family comprising > 10 enzymes. For many years it was believed that these enzymes catalyse protein breakdown unselectively, are highly redundant in their substrate specificity and are also expressed ubiquitously. This view has changed dramatically since a number of new lysosomal cysteine proteases with restricted expression and outstanding enzymatic activity have been described. In addition, knockout mice and selective protease inhibitors have been used to characterise specific functions of single proteases. In this review, some of these functions are discussed in relation to the lungs, especially the role of lysosomal cysteine proteases in matrix remodelling, immunoregulation and surfactant protein processing.

Cathepsin K--a marker of macrophage differentiation?

Cathepsin K is a cysteine protease with high matrix-degrading activity. Initially, cathepsin K was described as being expressed exclusively by osteoclasts. It was suggested that cathepsin K expression is a specific feature of cells involved in bone remodelling. The aim of this study was to investigate the hypothesis that cathepsin K is expressed not only in bone-resorbing macrophages, but also more generally in specifically differentiated macrophages, such as epithelioid cells and multinucleated giant cells in soft tissues. Specimens obtained from different organs and anatomical locations of patients suffering from sarcoidosis, tuberculosis, granulomas caused by foreign materials, and sarcoid-like lesions were investigated for the expression of cathepsins B, K, and L. Immunohistochemistry and in situ hybridization showed cathepsin K in epithelioid cells and multinucleated giant cells irrespective of the pathological condition and anatomical location, but not in normal resident macrophages. By immunoelectron microscopy, cathepsin K was discovered in cytoplasmic granules of multinucleated giant cells. In contrast, cathepsin B and cathepsin L were expressed ubiquitously in CD68-positive tissue macrophages, epithelioid cells, and multinucleated giant cells. The results demonstrate that cathepsin K, but not cathepsin B or cathepsin L, differentiates specific phenotypes of macrophages independently of the anatomical site. Its enzymatic characteristics, particularly its high matrix-degrading activity, suggest that cathepsin K-positive epithelioid cells and multinucleated giant cells are characterized by an enhanced specific proteolytic capability.

A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti.

During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages.

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

Feedback control of a master bacterial cell-cycle regulator.

The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.

The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle.

In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression. Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.

Regulation of a heat shock sigma32 homolog in Caulobacter crescentus.

High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase sigma32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in sigma32 levels after a temperature shift from 30 to 42 degrees C similar to that described for E. coli. In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30 degrees C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and sigma32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to sigma32 expression in this organism. Both the rpoH gene and sigma32 protein were expressed constitutively throughout the cell cycle at 30 degrees C. The isolation of rpoH provides an important tool for future studies of the role of sigma32 in the normal physiology of C. crescentus.

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Regulation of the ontogenic and regional expression of intestinal aminooligopeptidase.

Aminooligopeptidase (AOP) is the predominant peptidase in the small intestinal epithelium. During postnatal development in the rat, characteristic ontogenic and regional patterns of AOP activities become established. To investigate the molecular mechanisms regulating the maturational changes in AOP activity, we compared AOP synthesis and assembly in the jejunum of suckling (14-day-old) and weaned (28-day-old) rats. AOP synthesis was assessed in vivo by intraperitoneal labeling with [35S]methionine. Both AOP activity and AOP synthesis doubled at weaning, while its posttranslational processing remained unaltered. To examine the mechanisms responsible for generating the regional differences in AOP activity in weaned rats, we contrasted the de novo synthesis, kinetic properties, total immunoreactive protein, and degradation of the jejunal and ileal peptidases. Although AOP catalytic activity was significantly greater in the jejunum than in the ileum, its synthesis rate and substrate affinity (Km) were identical at both sites. However, the ileal peptidase was degraded more rapidly than the jejunal enzyme (t1/2 = 4 and 10 h, respectively). In summary, our data show that increased synthesis accounts for the ontogenic rise in intestinal AOP activity but that altered enzyme turnover produces the jejunal-ileal gradient in AOP activity in weaned rats. Thus the ontogenic and regional expressions of intestinal AOP are defined by transcriptional/translational and posttranscriptional regulatory mechanisms, respectively.

Ontogeny of intestinal lactase: posttranslational regulation by thyroxine.

To assess the molecular mechanisms underlying the regulation of lactase ontogeny by thyroxine (T4), we performed an in vivo study of lactase catalytic activity, synthesis, subunit structure, degradation, and enterocyte migration rates in propylthiouracil-induced hypothyroid rat pups, hypothyroid pups injected with T4, and normally weaned rats. Although lactase catalytic activity remained elevated in the hypothyroid rats and declined normally in the other two groups, lactase synthesis was constant among the groups. Lactase subunit structure was identical in normally weaned and T4-injected animals, but the 100-kDa moiety, characteristic of weaned rats, was absent in the hypothyroid pups. The turnover of lactase enzyme was more rapid in euthyroid and T4-injected rats than in hypothyroid animals (t1/2 = 17, 20, and 30 h, respectively). In addition, enterocyte migration was accelerated in the T4-injected rats and reduced in the hypothyroid group compared with controls. However, transit rate was not directly related to lactase activity. Our results suggest that T4 regulates lactase ontogeny by posttranslational mechanisms that include altered processing and increased degradation of the lactase enzyme.

Interactions of vitamin D with sodium zeolite A in rats from low and adequate vitamin D colonies.

Weanling rats (21 days old) from either a low vitamin D colony or an adequate vitamin D colony fed either a vitamin D adequate or a vitamin D deprived AIN-76A purified diet were used as a model to investigate interactions of vitamin D status with dietary sodium zeolite A (100 mg/kg body wt/day). Rats with adequate or replete vitamin D status had greater overall body weight gain, dry tibia weights and femur densities and ash weights than rats fed a vitamin D deprived diet. Addition of sodium zeolite A to the diets did not have an effect on plasma calcium, body weight gain or on femur density, ash and percent ash. Dietary sodium zeolite A increased total tibia fat in rats fed the vitamin D adequate diet and decreased total tibia fat in rats fed the vitamin D deprived diet. This effect of sodium zeolite A appeared to be beneficial to bone status in the groups fed adequate vitamin D, since these treatment groups had higher, although not significant, dry tibia weights with and without fat.

Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine.

Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme.

Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone.

During the third week of postnatal life, dramatic ontogenic changes occur in the morphology and enzymology of the small intestine of the infant rat, enabling the animal to make the transition from milk to solid food. To investigate the roles of T4 and GH in regulation of these changes, infant rats were hypophysectomized on day 6 of life by the transauricular technique. Hypophysectomy resulted in diminution of somatic and intestinal growth as well as abnormal maturation of the disaccharidases lactase, sucrase, and maltase when measured on day 25. Administration of either T4 or GH to hypophysectomized animals resulted in moderately increased intestinal growth, while complete restoration of small intestinal growth resulted from administration of the combination of both hormones. Although T4, GH, or the combination of hormones reduced lactase activities, T4 alone produced normal maturation of sucrase and maltase. Neither hypophysectomy nor hormone replacement affected aminooligopeptidase. The molecular structure of lactase, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was not altered to a major degree in hypophysectomized animals or animals that received hormone replacement, but minor alterations were evident in sucrase structure in hypophysectomy. These studies indicate that 1) T4 and GH actively participate in postnatal regulation of small intestinal ontogeny; 2) thyroid hormones act directly on developing intestinal tissues to independently produce the normal maturation of the disaccharidases by mechanisms that are not likely to involve alterations in processing of the enzyme-protein; and 3) maturation of aminooligopeptidase is not regulated by pituitary hormones, in distinct contrast to the disaccharidases.

Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation.

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.

Affinity labelling of the folate-binding protein in pig intestine.

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.