Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

黔桂边境六县植物区系组成及其特点

黔桂边境六县植物区系有维管束植物203科921属2255种(变种)。其中种子植物含25种以上的有22科,代表成分有樟科、山茶科、壳斗科、五加科、桑科和椴树科。区系的特点是:地理成分复杂、分布交错;热带、亚热带性质明显;区系起源古老;特有珍稀种类多;喜酸成分较喜钙成分占优势;草本与木本种类近等。此外,对本区系自然条件及植物资源利用也作了简述。     

用染色体原位杂交技术定位RFLP探针Fr.3-42于16p13

限制性片段长度多态性(RFLP)探针Fr.3-42(第九届人类基因定位国际会议编号D1(?)S21)为一长1.9kb的人类单拷贝EcoRI/HindⅢ片段,本实验采用染色体原位杂交方法,将该探针定位于16号染色体短臂末端(p13)。在另一研究中已证实Fr.3-42与人α-珠蛋白基因紧密连锁。     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Post-castration rebound of an androgen regulated prostatic gene

Summary After castration, the rat dorsolateral prostate M-40 mRNA initially decreased then rebounded to precastrated levels. The cellular site of M-40 expression and its renewed expression after castration was defined by in situ hybridization histochemistry. In situ hybridization with either a ³²P-labeled or biotin-labeled M-40 cDNA probe demonstrated that M-40 mRNA levels were higher in the lateral than dorsal prostate. A second androgen regulated gene, RWB, also was highly expressed in the lateral prostate. The biotinylated cDNA probes provided microscopic resolution of the expressing cells, revealing two distinct morphologies of lateral epithelium which expressed both the M-40 and RWB mRNA. These morphologies appeared in ducts which contained either epithelial cell sheets that were highly convoluted or thinner epithelial cells with a minimal degree of convolution. The RWB mRNA decreased in both cell populations in response to androgen withdrawal. The decline and reappearance of M-40 mRNA also appeared in both epithelial cell types. These data demonstrated that after castration the M-40 mRNA initially decreased as expected for an androgen sensitive gene and then progressed to a fully inducible state. The mechanism of this progression remains to be elucidated.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.