Urotensin II, a peptide hormone from the caudal neurosecretory system of the teleost, Gillichthys mirabilis, was isolated by using classical chromatographic techniques and high-performance liquid chromatography (HPLC). Direct microtechniques for sequence determination were used to establish its structure. Urotensin II from Gillichthys is a 1363-dalton dodecapeptide with the amino acid sequence AlaGly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. This sequence is homologous with somatostatin in positions 1 and 2 and 7-9. The sequence has been verified by the production of a bioactive synthetic urotensin II. The possible chemical and physiological significance of its homology to somatostatin is discussed.

The marrow microenvironment is a complex, three-dimensional structure composed of many cell types and abundant extracellular matrix. Much of the data are derived from analysis of the adherent layer of murine and, especially, human long-term marrow cultures. An essential feature of this in vitro counterpart to the marrow microenvironment is the presence of flat angulated cells functionally defined as marrow stromal cells with the following phenotype: type IV collagen(+), laminin(+), vimentin(+), CD10(+), muscle actin(+), Stro-1(+), and negative for CD45, Mac-1, and HLA-DR. Stromal precursors are Stro-1(+) and CD34(+). Regulation of hematopoietic precursors by the microenvironment occurs by elaboration of regulatory molecules such as hematopoietic cytokines, by cell-cell contact via adhesion molecules such as alpha 4 beta 1 integrin, and by interactions with components of the extracellular matrix as in the case of the glycosaminoglycan hyaluronic acid with cell-associated CD44. Although little about the regulation of stromal cell development itself is known, several studies indicate the transplantability of marrow stromal cells under specific conditions. These developments suggest a potential role of stromal cells in cell therapy. Transfected stromal cells may serve as suitable vehicles for gene delivery to correct single gene disorders in which the product of the target gene does not require stringent regulation as, for example, in the correction of Factor VIII and Factor IX deficiency. Further studies are warranted to investigate marrow stromal cell physiology and regulation to better understand hematopoiesis and to explore the possible use of stroma in therapy.

In tissue cultures of normal adult and malignant mammalian cells, homocystine has been substituted for methionine in a medium rich in folic acid and cyanocobalamin. Normal adult cells thrive. Three highly malignant cell types from three different species, including man, die.A metabolic defect seen to date only in cancer cells was uncovered during an investigation of transfer RNA methylation using tissue cultures of normal adult and malignant cells (1,2). In several lines of malignant cells we have observed an inability of the cells to survive and grow when methionine is replaced in the medium with homocystine. However, normal adult cell lines derived from mice, rats, and humans survive and grow well with this substitution. The apparent absolute dependence of the malignant cells on preformed methionine is of considerable theoretical interest but may also be of practical importance because it may offer another avenue for treating neoplastic disease.An earlier study (3), confirmed in our laboratory, has clearly indicated that dietary depletion of methionine has an inhibitory effect on the growth of Walker-256 carcinosarcoma. We became interested in this problem through our studies on methylation of transfer RNA in Walker-256 cells, and the present work demonstrates the dependence of this and other malignant cell lines on preformed methionine. In contrast to the malignant cell lines, we show that a number of normal adult cell lines can satisfy their methionine requirements with homocystine in a folic acid-, cyanocobalamin-rich medium. METHODSTissue cultures of Walker-256 (W-256), breast carcinosarcoma of rats; L1210, lymphatic leukemia of mice; J111, monocytic leukemia of humans; as well as liver epithelial and liver fibroblasts of rats, skin fibroblasts of mice, and fibroblasts from human breast and prostate (all concurrently PPLO negative) were grown in a commercially available, specifically modified McCoy's medium. The composition of the medium was routinely checked by amino-acid analysis before use. It was devoid of bactopeptone but reinforced with 15% fetal-calf serum that had been dialyzed for 24 hr at 40 against three changes of a solution containing 8 g of NaCl, 0.4 g of KCl, 1 g of glucose, and 0.35 g of NHCO3 per liter. In addition, the medium contained trace metals (10 ml/liter of medium of a solution containing FeSO4, 1 mg; CuCl2, 100 ,pg; ZnSO4, 100 jAg; MnCl2, 100 pg/liter) vitamin B12, 2-4 mg/liter; folic acid, 5-10 mg/liter; and either gentamycin (50 p&g/mi) or streptomycin sulfate (100 ug/ml) and penicillin 1133 G 100 U/ml. Except for subtractions or additions of methionine and homocystine, there were no other alterations in the medium. Two growth curves of each cell type were run simultaneously. One medium contained L-methionine in the usual concentration (15 mg/liter), while the other medium was devoid of methionine, but instead contained either L-homocystine (15 mg/liter) or DL-homocystine (30 mg/liter). Various amounts of vitamin B12, folic acid, homocystine, homocysteine, or the methyl ...

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) MATERIALS AND METHODS Mice. Female SJL/J mice were purchased from The Jackson Laboratory and were used between 8 and 12 weeks of age.Antigens. Peptides of MBP and PLP corresponding to amino acids 1-14, 91-103, and 139-151 were synthesized by solid-phase 9-fluorenylmethoxycarbonyl (FMOC) procedures. Peptides were purified by reverse-phase HPLC and were characterized by both HPLC and mass spectroscopy.Purification of dass H MHC. I-Al protein was purified from a Nonidet P-40 extract of spleen cell membranes from SJL/J mice by affinity chromatography using the monoclonal antibody 10-2.16 (specific for I-Ak and I-Al), coupled to Sepharose 4B beads. Extracted lysate from the high-speed (100,000x g) membrane fraction in a buffer of 10 mM Tris HCI, pH 8.3/0.5% Nonidet P-40/0.1 M sodium chloride/5 mM EDTA/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride was recycled over the preequilibrated column at 40C for 16 hr. The column was washed first with 10 bed volumes of deoxycholate buffer/10 mM Tris-HCl, pH 8.3/0.5% deoxycholate/0.1 M sodium chloride/5 mM EDTA/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride and then by 5 bed volumes of phosphate-buffered saline (PBS)/1% 1-octyl P-Dglycopyranoside buffer. Finally, I-AS was eluted with 20 mM phosphate buffer, pH 11/0.1 M sodium chloride/1% octyl glucoside/0.02% sodium azide/1 mM phenylmethylsulfonyl fluoride. Fractions were neutralized with acetic acid to a final Abbreviations: EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; PLP, proteolipoprotein; MHC, major histocompatibility complex. tTo whom reprint requests should be addressed. 11465The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.