Human hepatocellular carcinoma-associated changes of glycosphingolipids detected by two monoclonal antibodies FH2 and IB9

The changes of minor glycosphingolipids associated with human hepatocellular carcinomas were studied using monoclonal antibodies. Glycosphingolipids of twelve patients were isolated from the tumor tissues and their adjacent cirrhotic liver tissues. The immunoreactivities of glycosphingolipids with two monoclonal antibodies FH2 and IB9, that define x-structure (Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialosyl alpha 2-6galactosyl residue, respectively, were studied by a solid phase enzyme-linked immunosorbent assay and a chromatogram immunobinding assay. X-structure was detected in the upper-phase neutral glycosphingolipids from both the cirrhotic liver tissues and the tumor tissues, but was generally expressed more strongly in the latter tissues. The thin-layer chromatography patterns of x-active glycosphingolipids were more complex in the tumor tissues. Sialosyl alpha 2-6galactosyl residue was detected in two ganglioside fractions. One ganglioside with faster migration on a thin-layer chromatogram was found in both the non-tumorous tissues and the tumor tissues. Another ganglioside with slower migration was more often detected in the tumor tissues. It was concluded that some of minor glycosphingolipids detected by monoclonal antibodies FH2 and IB9 could be the 'tumor-associated' antigens.     

Potential enhancement of osteoclastogenesis by severe acute respiratory syndrome coronavirus 3a/X1 protein

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a lung disease with high mortality. In addition, osteonecrosis and bone abnormalities with reduced bone density have been observed in patients following recovery from SARS, which were partly but not entirely explained by the short-term use of steroids. Here, we demonstrate that human monocytes, potential precursors of osteoclasts, partly express angiotensin converting enzyme 2 (ACE2), a cellular receptor of SARS-CoV, and that expression of an accessory protein of SARS-CoV, 3a/X1, in murine macrophage cell line RAW264.7 cells, enhanced NF-κB activity and differentiation into osteoclast-like cells in the presence of receptor activator of NF-κB ligand (RANKL). Furthermore, human epithelial A549 cells expressed ACE2, and expression of 3a/X1 in these cells up-regulated TNF-α, which is known to accelerate osteoclastogenesis. 3a/X1 also enhanced RANKL expression in mouse stromal ST2 cells. These findings indicate that SARS-CoV 3a/X1 might promote osteoclastogenesis by direct and indirect mechanisms.     

Calpain activates two transglutaminases from porcine skin

Summary Two transglutaminases (TGase) with estimated molecular weight of 55,000 (55-K TGase) and 120,000 (120-K TGase) were partially purified from the cytosolic fraction of porcine skin (epidermis-rich preparation) using DEAE-cellulose and gel-filtration chromatographies. The enzyme activities of both trans-glutaminases were enhanced more than 20-fold by treatment with calpain (Ca²⁺-dependent cysteine proteinase) in the presence of Ca²⁺, and this enhancement was inhibited by adding EDTA, leupeptin, or an endogenous calpain-specific inhibitor protein (calpastatin). 55-K TGase was effectively activated by a smaller amount of calpain than was 120-K TGase, while known activating reagents such as thrombin and dimethyl sulfoxide or heat treatment preferentially activated 120-K TGase. One of the physiological functions of calpain in the epidermis may be the activation of epidermal transglutaminases.     

Production of monoclonal antibody to human esophageal cancer cell line

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen.     

Purification and characterization of calpains from pig epidermis and their action on epidermal keratin

Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.     

Carbohydrate auto-antigens in auto-immune hemolytic anemia

Anti-erythrocyte antibodies which appear in the sera of patients with auto-immune hemolytic anemia frequently recognize carbohydrate auto-antigens. Most of cold agglutinins are known to recognize the Ii-antigens, and Donath-Landsteiner antibodies which appear in patients with paroxismal cold hemoglobinuria are almost exclusively directed to the P-antigen. These carbohydrate auto-antigens are strongly expressed in various tissues and organs other than erythrocytes, and behave as differentiation- or developmental-antigens, in both humans and mice. The study of nucleotide sequences of human and murine anti-Ii antibodies shows that these antibodies share a highly homologous antigen-binding site in their VH regions. These results indicate that the carbohydrate auto-antigens in autoimmune hemolytic anemia are evolutionally conserved developmental antigens, and suggest that the immunoglobulin genes which encode variable regions of the auto-antibodies directed to these antigens are also conserved evolutionally.     

Immunological and virological status of a hemophiliac infected with human T cell lymphotropic virus type 1 and human immunodeficiency virus type 1, and results of therapy

The immunological and virological status of three hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) was monitored for 11 months. One of these patients was also infected with human T cell lymphotropic virus type 1 (HTLV-1), and HIV-1 could be isolated only from this patient among the three subjects. The doubly infected subject had the fewest T4 (helper) lymphocytes and the highest proportion of T8 lymphocytes with DR human leukocyte antigens (DR-Ag). The serum level of HIV-1 antigen increased in this patient during the observation period, and this increase was accompanied by a decrease in the proportion of DR-Ag-positive cells among the T8 lymphocytes. This patient was treated with 800 mg of zidovudine daily for 50 days. With treatment, the nonspecific clinical symptoms improved and the proportions of DR-Ag positive cells among the T8 lymphocytes decreased. Serum levels of HIV-1 antigen decreased immediately when therapy was started but later increased during therapy. In persons infected with both HTLV-1 and HIV-1, HIV-1 seems to proliferate readily.     

Carnitine: a neuromodulator in aged rats

A wide range of morphological and biochemical changes occur in the central nervous system with increasing age. L-carnitine, a naturally occurring compound, plays a vital role in fatty acid transport across the mitochondrial membrane. L-carnitine (300 mg/kg body wt/day) was administered intraperitoneally to young and old male Wistar rats for 7, 14, and 21 days. Carnitine, dopamine, epinephrine, and serotonin levels were assayed in discrete regions of the brain. Carnitine supplementation increased the levels of dopamine, epinephrine, and serotonin in the experimental animals in our study. Response to carnitine supplementation varied among the brain regions that have been studied. The regions rich in cholinergic neurons such as the cortex, hippocampus, and striatum showed more response after 21 days of carnitine treatment. The results of the present study suggest the role of L-carnitine as a neuromodulator and antiaging medication.