The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Comparison of patterns of hybrid resistance to the BALB/c plasmacytomas LPC-1 and MPC-11

Summary Patterns of genetic control of hybrid resistance to the BALB/c plasmacytoma LPC-1 were studied for comparison with those to MPC-11, a plasmacytoma investigated previously. The overall patterns of hybrid resistance to the two tumors were similar, i.e., hybrids between BALB/c and BALB congenic resistant (CR) strains, A and A CR strains, SJL and DBA/2 were as susceptible to LPC-1 as BALB/c mice themselves, whereas hybrids between BALB/c and AKR, C57BL/Ks, DBA/1, C57BL/6 (B6), C57BL/10 (B10) and B10 CR strains were resistant to LPC-1 as previously shown with MPC-11. Heterozygosity within the H-2 complex alone was insufficient for resistance to either tumor. Among hybrids between BALB/c and the B10 CR strains, however, the presence of certain H-2 haplotypes influenced the degree of resistance seen and this H-2 effect was different for the two tumors. A sex effect on resistance to LPC-1, but not to MPC-11, was seen among F₁ hybrids between BALB/c and DBA/1 although not in any other F₁ hybrids. Among ((B10×BALB/c)F₁×BALB/c) and (BALB/c×(B10×BALB/c)F₁) and ((BALB/c×B10)F₁×BALB/c) and ((BALB/c×B10)F₁×BALB/c) backcross mice, however, significantly more males than females were resistant to LPC-1 and the results of this study are compatible with the idea that in F₁ hybrids between BALB/c and B10, resistance to LPC-1 is controlled by two dominant autosomal genes, one of which is sex-limited and neither of which is linked to H-2. In contrast, hybrid resistance to MPC-11 in this cross is controlled by a single gene. Cross-protection experiments indicated that the two tumors share at least one tumor-associated transplantation antigen.     

Some Properties of Thiosulfate-Oxidizing Enzyme from Marine Heterotroph 16B

Thiosulfate-oxidizing enzyme has been demonstrated in cell-free extracts of the marine, thiosulfate-oxidizing pseudomonad strain 16B. The enzyme, partially purified by ion-exchange chromatography and calcium phosphate gel treatment, catalyzed the oxidation of thiosulfate to tetrathionate with the concomitant reduction of ferricyanide. Native but not mammalian cytochrome c was also reduced by the enzyme in the presence of thiosulfate. The enzyme was located exclusively in the supernatant of ultracentrifuged cell extracts. The most purified enzyme preparation, like intact cells, exhibited a temperature optimum of 30 to 31°C. However, it exhibited no definite pH optimum. At pH 6.1 to 6.3 and 30°C, the K_m for thiosulfate was 1.57 mM. At lower temperatures, the apparent K_m for thiosulfate increased, but the apparent maximum velocity remained virtually unchanged. Thiosulfate oxidation in intact cells exhibited an increase in the pH optimum at lower temperatures. The thiosulfate-oxidizing enzyme of marine heterotroph 16B is compared with thiosulfate-oxidizing enzymes from other bacteria, and the effect of temperature on the relationship between pH and thiosulfate oxidation is discussed with reference to the natural habitat of the bacterium.     

Effect of mycorrhizal inocula on the growth of Sitka spruce seedlings in different soils

Summary Different mycorrhizal fungi were tested for their effectiveness in promoting growth of Sitka spruce seedlings, in two contrasting soils, in a glasshouse pot experiment. In nursery soil,Laccaria amethystina significantly improved growth of seedlings in comparison toL. laccata. Seedlings inoculated with a forest isolate ofThelephora terrestris were significantly larger than those inoculated with a nursery isolate when grown in forest soil. The effectiveness ofComplexipes moniliformis in forest soil was poor in comparison to other mycorrhizal fungi. Strains aswell as species of mycorrhizal fungi affect seedling growth differently. These effects are further influenced by soil type.     

Specific and nonspecific T-cell recruitment in viral meningitis: Possible implications for autoimmunity

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2⁺ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.     

The enzymic estimation of glutamate and glutamine

A method of estimating glutamic acid is described, based on its dehydrogenation by glutamate dehydrogenase coupled, by means of N-methylphenazine methosulphate, to the reduction of tetrazolium salts. The method is suitable for the estimation of 0-0.3mumole of glutamic acid. The response is linear, but not stoicheiometric: possible reasons for this are discussed. If suitable precautions are taken, the use of a partially purified preparation of glutaminase makes it possible to estimate glutamine also.     

Unrestricted recognition of a nonpeptide antigen by CD8+ cytolytic T lymphocytes

CD8+ murine CTL that are specific for an unusual nonpeptide Ag, the heme moiety of hemoglobin, have been derived by in vitro stimulation of spleen cells with hemin. Such CTL demonstrate a requirement for the expression of class I Ag on target cells, yet appear to be unrestricted to the extent that both syngeneic and allogeneic targets precoated with hemin are sensitive to lysis. A series of CTL clones with specificity for hemin was derived from C57BL/6 mice. They exhibited the same type of promiscuous recognition that was observed in CTL populations from a number of different strains. The possibility that hemin acts as a nonspecific mediator of lysis by CTL was ruled out by the fact that a variety of CTL populations and clones specific for different Ag did not exhibit hemin-specific lysis. Some explanations offered to explain these results include 1) the possibility that hemin is recognized by binding to a site on the MHC other than the Ag-binding groove, and 2) the possibility that TCR recognition of a rigid molecule, such as hemin, may be less sensitive to polymorphic variation in the MHC than is recognition of a conventional peptide Ag whose conformation may differ significantly when bound to MHC molecules whose sequences differ within the Ag-binding groove.