Cytotoxic action of 2-deoxy-D-glucose tetraacetate upon human lymphocytes, fibroblasts and melanoma cells

Obesity can play a significant role in chronic diseases, sudden unexpected death, and morbid obesity may be important as a cause of death for forensic pathologists. Our study attempted to determine if there is a correlation between panniculus measurements and body mass index (BMI) since BMI has been used in most studies to categorize obesity. Using data obtained from a review of 524 adult autopsies conducted at the University of Michigan from 1990 to 1992 we were able to show a correlation between both thoracic and abdominal panniculus and BMI (r2 = 0.335 and 0.296 respectively) which is statistically significant (p = 10(-47) and 10(-41) respectively). A prospective study confirmed the correlation (r2 = 0.552 for thoracic and 0.436 for abdominal panniculus) when the measurements were taken at the xyphoid process and 3 cm below the umbilicus. Using these data we calculated a panniculus index (PI) which is equal to the thoracic + abdominal panniculus in centimeters divided by the square of the height (in meters). The PI strongly correlated with BMI and was able to predict obesity. Using a BMI cutoff of 39 for morbid obesity, a PI value of 4.07 for females and 3.25 for males predicted morbid obesity with the probability of a false positive less than or equal to 2.5%. Mild and severe obesity could also be determined using the PI. Based on these data we've concluded that a concise mathematical relationship does exist between BMI and panniculus measurements. Therefore panniculus measurements can be used either as a surrogate measurement of morbid obesity or to support BMI calculations.     

Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.     

Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas

We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%). On the basis of these structural characteristics, we have tentatively called this novel protein cathepsin L2. The cDNA encoding the mature cathepsin L2 was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated cDNA encodes a functional cysteine proteinase. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis. This finding is in marked contrast with the wide tissue distribution of most cysteine proteinases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis. Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carcinomas but not in normal colon or mammary gland or in peritumoral tissues. Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas. These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Utilization of the same DNA replication origin by human cells of different derivation

In the past, a highly sensitive and efficient method was developed to map DNA replication origins in human cells, based on quantitative PCR performed on nascent DNA samples. This method allowed the identification of a replication origin in the myeloid HL-60 cell line, located on chromosome 19 within an approximately 500 bp segment near the lamin B2 gene [Giacca et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 7119]. The same procedure has now been further simplified and extended to a variety of other exponentially growing human cells of different histological derivation (three neural, one connectival and one epithelial), with a nearly diploid chromosomal content. In all the six cell lines tested, the origin activity within the lamin B2 gene domain was localized to the same region. Furthermore, the lamin B2 origin was also found to be active in stimulated, but not in quiescent, peripheral blood lymphocytes.     

Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.     

Glycosyltransferase activities in cultured endothelial cells of bovine brain microvascular origin

We report two cases of salmonella osteomyelitis isolated to the pelvis in white adolescents aged 12 and 16 years. No underlying medical condition predisposed these children to salmonella osteomyelitis, and the clinical course was prolonged before definitive diagnosis. The key to diagnosis and the localization of the site of the pathologic condition was made from radionuclide studies performed 2-3 weeks from the onset of symptoms. Clinicians should be aware of isolated salmonella osteomyelitis of the pelvis in normal children, especially when imaging studies are normal at initial presentation. Technetium-labeled bone scans may be normal < or = 2 weeks from the onset of symptoms. Definitive diagnostic testing should include a gallium scan and computed tomography scan when technetium bone scans are negative. Treatment with antibiotics alone is successful.     

Lymphopain, a cytotoxic T and natural killer cell-associated cysteine proteinase

Through differential screening of established human leukaemia cell lines, we have identified and molecularly cloned lymphopain, a novel cysteine proteinase of the papain family. Lymphopain exhibits a remarkably restricted cellular pattern of expression, being predominantly expressed in cytotoxic T-lymphocytes and natural killer cells. The human lymphopain locus maps to chromosome 11q13, encodes a polypeptide of 376 amino acids and is conserved in the mouse. Both human and murine forms appear more closely related to protozoan papain-like enzymes than to other mammalian members of the papain family. The cellular distribution of lymphopain expression, together with the functional demonstration of lymphopain-associated proteinase activity in vitro, is suggestive of a role for lymphopain in immune cell-mediated, cell killing.     

Adenosine receptor mediates motility in human melanoma cells

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.     

Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.     

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain)

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD(or E)AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.     

Involvement of a lysine-specific cysteine proteinase in hemoglobin adsorption and heme accumulation by Porphyromonas gingivalis

The oral anaerobic bacterium Porphyromonas gingivalis, a major pathogen of advanced adult periodontitis, produces a novel class of cysteine proteinases in both cell-associated and secretory forms. A lysine-specific cysteine proteinase (Lys-gingipain, KGP), as well as an arginine-specific cysteine proteinase (Arg-gingipain), is a major trypsin-like proteinase of the organism. Recent studies indicate that the secreted KGP is implicated in the destruction of periodontal tissue and the disruption of host defense mechanisms. In this study, we have constructed a KGP-deficient mutant to determine whether the cell-associated KGP is important for pathophysiology of the organism. Although the mutant retained the strong ability to disrupt the bactericidal activity of polymorphonuclear leukocytes, its hemagglutination activity was reduced to about one-half that observed with the wild-type strain. More important, the mutant did not form black-pigmented colonies on blood agar plates, indicating the defect of hemoglobin adsorption and heme accumulation. Immunoblot analysis showed that the expression of a 19-kDa hemoglobin receptor protein, which is thought to be responsible for hemoglobin binding by the organism, was greatly retarded in this mutant. The mutant also showed a marked decrease in the ability to degrade fibrinogen. These results suggest the possible involvement of KGP in the hemoglobin binding and heme accumulation of the organism and in the bleeding tendency in periodontal pockets.     

Variable expression of Mn SOD in three different human melanoma cell lines

The mechanism of age-related cortical bone loss was investigated in 229 Japanese women, 41-94 years of age, by metacarpal bone mass measurement. While no significant correlation was found between bone width and age, a significant increase in bone marrow width, and significant decreases in cortical bone density and total bone mass were observed in association with aging (P < 0.0001). There was a significant negative correlation between total bone mass and bone marrow width (r = -0.239; P < 0.0005), and significant positive correlations between both total bone mass and cortical bone density (r = 0.539; P < 0.0001) and cortical bone width (r = 0.839; P < 0.0001). The findings suggested that age-related cortical bone loss in middle-aged and elderly women resulted from two different factors; a decrease in cortical bone density caused by progression of intracortical porosity, and a decrease in cortical bone width as a result of bone loss on the endosteal surface. The latter had a greater influence on an age-related cortical bone loss than the former.     

Alterations in gene expression and signal transductions in human melanocytes and melanoma cells

The development of techniques to cultivate human primary melanocytes in vitro has provided the technical foundation for understanding the biology of this cell. Human melanocytes require various growth factors and agents for proliferation in vitro. These compounds activate two major signal transduction pathways: a calcium- and phospholipid-dependent (protein kinase C or PKC) pathway and a cyclic AMP (cAMP)-dependent (protein kinase A or PKA) pathway. Alterations in these signal transduction pathways coupled with changes in specific genes (protooncogenes, growth factors, and tumor suppressor genes) have been observed in human melanoma cells compared with normal melanocytes. Our own work indicates that loss in the expression of the PKC beta II isotype is a common, if not universal, alteration that occurs early in human melanocyte transformation. In this review, we concentrate on alterations in the signal transduction pathways in human melanocytes and melanoma cells and delineate how an understanding of these changes may allow us to understand the molecular mechanisms involved in human melanocyte transformation.     

Effect of bile salts on lactosylceramide beta-galactosidase activities in human brain, liver and cultured skin fibroblasts

The effect of bile salts on the hydrolysis of lactosylcermide by human beta-galactosidases in vitro was studied using cultured skin fibroblasts, liver and brain tissue. The evidence for two distinct enzymes that can catalyze the hydrolysis of lactosylceramide was observed when the bile salt was changed from pure sodium taurocholate to either crude taurocholate, or pure glycodeoxycholate, taurodeoxycholate or taurochenodeoxycholate. Tissues from patients with Krabbe's disease were found to be deficient in lactosylceramide beta-galactosidase activity (lactosylceramidase I) when pure taurocholate was used in the assay. When crude taurocholate was used in the assay, the Krabbe patients appeared to have normal activity for this enzyme. In place of crude taurocholate the pure salts of glycodeoxycholate, taurodeoxycholate and taurochenodeoxycholate worked even better to stimulate the second lactosylceramide beta-galactosidase activity and GM1 gangliosidosis patients exhibiting little if any activity. Therefore, lactosylcermidase I is stimulated by crude taurocholate or pure glycodeoxycholate, taurodeoxycholate and taurochenodeoxycholate. The use of pure bile salts to assay lactosylceramidase I and II will result in better reproducibility for these enzyme activities between laboratories.