Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Biotransformation of diphenyl ether by the yeastTrichosporon beigelii SBUG 752

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.     

Crown alterations induced by decline: a study of relationships between growth rate and crown morphology in beech (Fagus sylvatica L.)

One of the first symptoms expressed by declining trees is reduced growth in stem diameter and length increment. The possibility of a relationship between length increment and crown thinning in beech (Fagus sylvatica L.) was investigated by developing a computer model to simulate first order branching patterns of the apical 2 m of monopodially branching beech trees, 70–100 years old, for a range of length increment rates. The model was based on values for branching angle, main axis and branch length increment, number of branches produced per year and branch mortality rates for six healthy and declining trees. Shoot growth rates in the apical 2 m of the sample trees ranged from about 5 cm/year (decline class 3) to 43 cm/ year (healthy). Simulations of branching patterns in the apical 2 m of trees growing at different rates indicated that, when growth rate exceeded about 20 cm/year, total first order branch length and area explored were independent of growth rate. When growth rates fell below this value there was a reduction in total area explored and first order branch length due primarily to the formation of fewer branches. More acute branching angles contributed to a reduction in the area explored. Growth rate-related crown thinning could increase the risk of bark necrosis and secondary pathogen infection during dry and/or hot spells.     

A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins

Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Ville de Laval, Québec H7N 4Z3, Canada.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.     

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase,a thermostable serine protease fromThermoactinomyces vulgaris

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with K_i values of 7×10^–8 M and 2×10^–7 M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.     

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.     

Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physicochemical exposures

Dictyocaulus viviparus larvae were exposed to ox bile and CO₂ at intervals during their cultivation to the infective stage. Preinfective and young infective larvae were stimulated by CO₂. Bile slightly inhibited preinfective larvae, but stimulated the infective stage. Old coiled, resting infective larvae were stimulated by bile down to a concentration of 10 ppm of bile dry matter, by vertebrate biles of pig, sheep, newborn calf, cow, guinea pig, dog, and chicken, as well as by defatted bile dry matter and by glyco-, tauro-, glycodeoxy-, and taurodeoxycholates. Continuous bile exposure appeared necessary to maintain high larval activity. A high pCO₂ as well as a low redox potential potentiated the effect of bile, but had no effect alone. Exposure to pepsin-HCl and to trypsin had only a minor stimulatory effect.