Antibody responses to early antigens of varicella-zoster virus (VZV) during varicella and zoster

The antibody responses to membrane and early antigens and thymidine kinase of varicella-zoster virus (VZV) were studied in sera during both varicella and zoster by a test with fluorescent antibody to membrane antigen (FAMA), staining the biochemically transformed cells by the immunofluorescent technique and neutralization of virus-specific thymidine kinase activity, respectively. Similar increases in FAMA antibody titers were demonstrated in sera from patients with varicella and zoster. IgM was detected in both groups, but appeared earlier during varicella than during zoster. Furthermore, the antibody titers to early antigens and virus-specific thymidine kinase were higher in patients with zoster than in those with varicella. These data suggest that different types of antibody responses occur during varicella and zoster.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease

Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Xyloglucan in the Cell Walls of Suspension-Cultured Rice Cells

The xyloglucan present in the 24% KOH extract of the cell wallsof suspension-cultured rice cells was characterized by fragmentationanalysis with Trichoderma viride cellulase and Aspergillus oryzaeß-D-glucosidase. The xyloglucan is composed mainlyof the following oligosaccharide units: Results showed that the xyloglucan of suspension-cultured ricecells is more extensively branched than is that of rice seedlings.Another structural characteristic of the former xyloglucan isthe presence of D-galactosyl-D-xylosyl side chains that arenot found in the latter. (Received June 15, 1984; Accepted January 11, 1985)