Activities evoked in different strain structures by stimulation of the contralateral tectum in the chick]

In the chick, as soon as hatching, the responses recorded in various areas in the "accessorius hyperstriatum" (Wulst) seem to be very localized in relation with the stimulated region of the contralateral tectum. Very similar results are obtained within the other striate structures. These observations suggest in first part, that the tectal efferents project very diffusely to various striate structures and, in other part, that it exists in these a rather precise tectotopy and a columnar organisation. These results appear very constant during the first month of the hatching.     

Evaluation of antibody inhibition assays of melanoma antigens in sera to monitor tumor growth in melanoma patients

Summary It was reported previously that melanoma leukocyte-dependent antibody (LDA) in the sera of melanoma patients was inhibited by small-molecular-weight (small-mol.-wt.) glycoproteins which were similar to cell surface antigens identified in cell membrane extracts of melanoma cells. The present study was to determine whether measurement of the levels of these factors in sera may be a useful monitor of tumor growth in melanoma patients. Small-mol.-wt. fractions were obtained by gel filtration or membrane chromatography of acidified sera and tested for their ability to inhibit LDA in ⁵¹Cr release cytotoxic assays. A panel of LDA was used, consisting of three antisera from melanoma patients, which appeared relatively specific for melanoma, and three non-melanoma antisera against carcinoembryonic antigen, ₂ microglobulin, and fetal antigens. The results showed that in patients with melanoma, approximately 70% had melanoma LDA-inhibitory activity detected in the small-mol.-wt. fractions of their sera when these were tested against the panel of melanoma LDA. The specificity of the inhibitory activity for melanoma LDA was shown by failure of the serum fractions to inhibit non-melanoma LDA and by absence of inhibitory activity in equivalent serum fractions from non-melanoma carcinoma patients for melanoma LDA. The levels of melanoma LDA-inhibitory activity in the serum fractions appeared to correlate with tumor growth, as shown by clearance of the inhibitory activity after surgical removal of melanoma and reappearance in the serum of patients who subsequently developed recurrent melanoma. The 30% false-negative rate indicated that the assays could not be used to reliably exclude melanoma, but the close correlation with tumor growth and the low number of false-positive results suggested that in 70% of patients detection of these small-mol.-wt. antigens would be of value to detect recurrence from melanoma and to monitor the effectiveness of therapy.     

Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: unique regulation modulated by multiple genetic sites.

Summary Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Val^r). The Val^r isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHAS^r), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHAS^r phenotype were found to be cotransducible with glyA. However, no mutations causing a Val^r phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Val^r linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHAS^s) and AHAS^r in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHAS^s, only, in the mutant containing ilv-662. Further, both AHAS^s and AHAS^r in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHAS^r synthesis in the strain carrying ilv-660 and only partially repressed AHAS^r in the strain carrying ilv-662. Unexpectedly, AHAS^r synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.     

Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.     

Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen.

Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.     

Comparison of structural requirements for interaction of the same peptide with I-Ek and I-Ed molecules in the activation of MHC class II-restricted T cells.

We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.