Glycoprotein Biosynthesis in Chlamydomonas II. No Evidence for Lipid Intermediates in Protein Galactosylation

A crude membrane fraction from Chlamydomonas reinhardii transferredradioactivity from UDP-[¹⁴C]galactose to endogenous lipids.Most of the radioactivity was detected in digalactosyl diacylglycerolmoieties in which only the distal galactose residue (-linkedto monogalactosyl diacylglycerol) was labeled. A second compoundwas identified as monogalactosyl diacylglycerol labeled in thegalactose moiety ß-linked to glycerol. In additionto these two galactolipid species, trace amounts of radioactiveglucose were detected in the aqueous phase after mild acid treatmentof the total lipid fraction. This demonstrates the presenceof a 4-epimerase and provides indirect evidence for the presenceof a small amount of polyprenyl-monophosphate-glucose whichwas presumably not detectable per se because of the bulk ofneutral galactolipids. The failure to detect polyprenyl-phosphate-galactoseor mild acid labile galactose at any time during the incubationsuggests that galactosylation of Chlamydomonas proteins mightoccur without the involvement of lipid intermediates. (Received May 10, 1982; Accepted August 21, 1982)     

Glycoprotein Biosynthesis in Chlamydomonas: I. IN VITRO INCORPORATION OF GALACTOSE FROM UDP-[C]GALACTOSE INTO MEMBRANE-BOUND PROTEIN

A crude membrane fraction from Chlamydomonas reinhardii was found to catalyze d-galactose transfer from UDP-galactose to endogenous proteins. Highest incorporation rates were achieved by incubation at 25 degrees C and pH 7.5 in the presence of 10 millimolar Fe(2+). Hydrolytic studies on the labeled polymer revealed that radioactivity was attached to protein via an alkali-stable and acid-labile linkage. Identification of galactose as the only labeled sugar in the acid hydrolysate and results of a tentative estimation of the molecular weight of the charged alkaline degradation product indicate that monomeric galactose units are transferred to form an O-glycosidic bond with peptidyl hydroxyproline. No indications were found for a similar linkage to serine which, in contrast to the hydroxyproline-O-glycoside linkage, is acid-stable but is cleaved by beta-elimination. Chromatography of the sodium dodecyl sulfate-solubilized polymer on Sepharose-6B demonstrated that galactosyl residues are mainly associated with proteins which are of considerably higher molecular weight than are the majority of sodium dodecyl sulfate-denatured membrane proteins in this fraction.     

IL-1β promotes hypoxic vascular endothelial cell proliferation through the miR-24-3p/NKAP/NF-κB axis

Purpose: Our previous data indicated that miR-24-3p is involved in the regulation of vascular endothelial cell (EC) proliferation and migration/invasion. However, whether IL-1β affects hypoxic HUVECs by miR-24-3p is still unclear. Therefore, the present study aimed to investigate the role and underlying mechanism of interleukin 1β (IL-1β) in hypoxic HUVECs.Methods: We assessed the mRNA expression levels of miR-24-3p, hypoxia-inducible factor-1α (HIF1A) and NF-κB-activating protein (NKAP) by quantitative real-time polymerase chain reaction (RT-qPCR). ELISA measured the expression level of IL-1β. Cell counting kit-8 (CCK-8) assays evaluated the effect of miR-24-3p or si-NKAP+miR-24 on cell proliferation (with or without IL-1β). Transwell migration and invasion assays were used to examine the effects of miR-24-3p or si-NKAP+miR-24-3p on cell migration and invasion (with or without IL-1β). Luciferase reporter assays were used to identify the target of miR-24-3p.Results: We demonstrated that in acute myocardial infarction (AMI) patient blood samples, the expression of miR-24-3p is down-regulated, the expression of IL-1β or NKAP is up-regulated, and IL-1β or NKAP is negatively correlated with miR-24-3p. Furthermore, IL-1β promotes hypoxic HUVECs proliferation by down-regulating miR-24-3p. In addition, IL-1β also significantly promotes the migration and invasion of hypoxic HUVECs; overexpression of miR-24-3p can partially rescue hypoxic HUVECs migration and invasion. Furthermore, we discovered that NKAP is a novel target of miR-24-3p in hypoxic HUVECs. Moreover, both the overexpression of miR-24-3p and the suppression of NKAP can inhibit the NF-κB/pro-IL-1β signaling pathway. However, IL-1β mediates suppression of miR-24-3p activity, leading to activation of the NKAP/NF-κB pathway. In conclusion, our results reveal a new function of IL-1β in suppressing miR-24-3p up-regulation of the NKAP/NF-κB pathway.     

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.     

Notes on the Orchid Flora in the Hengduan Mountain Region,China

The Hengduan Mountain Region on the south-eastern fringe of the Qinghai- Xizang (Tibet) Plateau is located in W. Sichuan, N. W. Yunnan and E. Xizang, with a wide area of juxtaposition from the east to the west, the mountains extending and the rivers flowing from the north to the south. In this paper it covers an area from Daojie, Wayao, Yingping, Yangbi, Dali of Yunnan and Dukou of Sichuan in the south, to Banbar, Dengqeu, Shenda of Tibet and Serxu, Dainkog, Shuajingsi and Nanping (Jiuzhaigou) of Sichuan in the north, and from Lharong, Baxoi and Zayü of Tibet in the west, to Maowen, Wenchuan, Mt. Erlang, Mt. Emei and Xichang of Sichuan in the east (Fig. 1.). The Gongga Mountain is the highest in the region, its summit being at an altitude of 7556m, whereas the Dadu River Valley in the eastern part of the area is only 1150 m above sea level. Therefore, the relative height is about 6400 m in the region. The Hengduan Mountain Region is well-known for its various topography, complex natural conditions and rich flora. The floristic composition and features of orchids in Hengduan Mountain Region. 1. The species of orchids are abundant in the region. As we know so far, orchids in the Hengduan Mountain Region comprise 91 genera and 363 species with 9 varieties, and thus it is one of concentration centres of orchids in China, making up 56.17% of the total number of orchids genera in China, only less than in Yunnan and Taiwan, and 34.87% of the total number of orchids species in China, only less than in Yunnan and Sichuan. 2. The orchids genera in the Hengduan Mountain Region are complex in geographical components as indicated below: (1) Four geneva are endemic to China and one of them is endemic to the region. (2) Fourteen genera are of the north temperate distribution pattern, 2 of the Old World temperate one, 18 of the East-Asian one (including Sino-Himalayan and Sino-Japanese) and 3 of the East-Asian-North American one. (3) Twenty one genera belong to the tropical Asian distribution pattern, 3 to the tropical Asian-tropical African one, 13 to the tropical Asian-tropical Australian one, 1 to the tropical Asian-tropical South American one, 8 to the Old World tropical one and 2 to the pantropical one. (4) Two genera are cosmopolitan. The analysis of genera: Fourty eight genera (containing 151 species with 4 varieties) of the tropical distribution occur in the region, among which Calanthe and Cymbidium distributed in the temperate region, and Bulbophyllum and Peristylus in the subtropical part of China are comparatively abundant (with over 10 species), but the other 25 genera are monospecific and 11 genera each contain only 2-3 species. Some epiphytic genera mainly distributed in tropical Asia and belonging to tropical florestic elements, such as Vanda, Luisia, Schoenorchis, Flickingeria, Monomeria, Kingidium, Acampe, Phalaenopsis, Thrixspermum, Eria, Taeniophyllum, and terrestrial genera, such as Aphyllorchis, Collabium, Mischobulbum, Paphiopedilum, Thunia, Brachycarythis, Satyrium, Corybas, Geodorum, Zeuxine, Tropidia, have the Hengduan Mountain Region as the northern limit of distribution. Of 151 species with 4 varieties, 41 species with 4 varieties are endemic to China, and 14 species with 3 varieties of them are endemic to the area, making up 3.86% of the total in the region under discussion. There are 41 genera (containing 189 species with 5 varieties) of the temperate distribution, which occur in the region. Among them Platanthera (22 species with 1 variety), Cypripedium (17 species), Herminium (16 species), Amitostigma (15 species with 1 variety), Orchis (12 species), Hemipilia (8 species with 1 variety), Neottianthe (4 species), Gymnadenia (4 species), Diphylax (3 species), Bletilla (3 species), have the Hengduan Mountain Region as the distribution centre and differentiation centre. Among the 189 species with 5 varieties, 111 species with 5 varieties are endemic to China, and 54 species with 5 varieties are endemic to the area, making up 14.88% of the total of orchids in the Hengduan Mountain Region. Although the number of temperate distribution genera is smaller than that of tropical distribution ones, several points may be mentioned: (1) The Hengduan Mountain Region is distribution centre and differentiation centre of a number of temperate genera in China, and is the northern limit of many genera mainly distributed in the tropics. (2) The number in the former category is obviously larger than that in the latter. (3) Endemic species in the former category in the area are over three times as many as those in the latter. The differentiation of species of the temperate distribution genera is obviously stronger than the tropical ones, which characterizes the orchid flora in the area as the temperate one. The life forms of genera. The orchid flora in the Hengduan Mountain Region so far known comprises 91 genera, among which 51 are terrestrial, 32 epiphytic and 8 saprophytic, thus with the terrestrial one dominant. The analysis of species: The orchid flora in the Hengduan Mountain Region so far known comprises 363 species with 9 varieties. Their distribution patterns and floristic components, to which they belong, are indicated as follows: (1) Fifty four species, belonging to 33 genera, are widespread, covering the whole East Asian Region, but 6 of them are endemic to China. (2) Forty four species, belonging to 27 genera, are the elements of the Sino-Japanese Subregion, but 22 species of them are endemic to China. (3) One hundred and ninety five species with nine varieties, belonging to 53 genera, are the elements of the Sino-Himalayan Subregion under discussion: (A) Four species (i.e. Aphyllorchis alpine, Listera divaricata, L. pinetorum and Oreorchis micrantha) are distributed in the Himalayan Region and S. E. Xizang (Tibet), western part of this region. (B) Twenty five species, belonging to 17 genera, are distributed in N. W. Yunnan and the Himalayan Region (Appendix, 1.). (C) Sixteen species, belonging to 11 genera, are distributed in the Himalayan region and W. Sichuan. Among them 6 species occur only with Mt. Emei as the easternmost limit and 10 species occur in the region west of Mt. Emei. (D) Ten species, belonging to 9 genera, are distributed in the Himalayan region, this region and S. Shaanxi, S. Gansu or S. E. Qinghai. (E) Eight species, belonging to 6 genera, are distributed in the Himalayan region and this region. Among them 6 species have their range extending eastwards to Guizhou and 2 species eastwards to Guangxi. (F) Five species, belonging to 5 genera, having their range extending from this region southwards to N. Burma. (G) One handred and twenty seven species with nine varieties are endemic to China behind discussion. (4) (A) Three species (i.e. Anoectochilus moulmeinensis, Bulbophyllum forrestii and Liparis chapaensis) are distributed in Indo-China, Burma and the region. (B) Nine species, belonging to 7 genera, are distributed in Indo-China, N. E. India and this region. (C) Forty six species, belonging to 21 genera, are distributed in Indo-China, the Himalayan Region and this region (Appendix, 2.). (D) Twelve species, belonging to 11 genera, are distributed in Indo-China and this region (Appendix, 3.) 3. The vicarism is obvious in the orchid flora of the Hengduan Mountain Region. There are 10 species-pairs (in genera Calanthe, Tropidia, Anoectochilus, Mischobulbum, Bulbophyllum, Gymnadenia, Pogonia, Tipularia, Tulotis, Orchis, etc.) of the horizontal vicarism and 7 species-pairs (in genera Epigeneium, Epipogium, Platanthera, Pogonia, etc.) of the vertical vicarism in the region. 4. The endemic species are prolific in the region. In the orchid flora of the Hengduan Mountain Region there are 155 species and 9 varieties endemic to China: (1) Six species are widespread in the whole East-Asian Region. (2) Twenty two species are the elements of the Sino-Japanese Subregion. (3) One hundred and twenty seven species with nine varieties are the elements of the Sino-Himalayan Subregion. Among them 69 species with 5 varieties are endemic to the region (Appendix, 4.), making up 19% of the total in the region; other 58 species with 4 varieties are distributed in the region and neighbouring regions or provinces of it (Appendix, 5.). 5. Remarkable differentiation of the orchid flora in the Hengduan Mountain Region is shown by evident vicarism and abundance of endemic elements, exampled by Amitostigma, Herminium, Orchis, Cypripedium, Platanthera, etc. and one group of Platanthera, which is confined to the south fringe of the Xizang (Tibet) Plateau-Hengduan Mountain Region. The group consists of 12 species, of which one (P. edgeworthii) is distributed in the Western Himalayas from Hazara in Pakistan to Kumaun in India, and all the other 11 species (i.e.P. stenantha, P. bakeriana, P. roseotincta, P. deflexilabella, P. longiglandula, P. exilliana, P. chiloglossa, P. leptocaulon, P. platantheroides, P. clavigera and P. latilabris) occur in China, with 3 of them (i.e.P. deflexilabella, P. longiglandula and P. chiloglossa) endemic to China. According to their structure of gynostemum and form of labellum they belong to Platanthera without question, although they are different from the other members of Platanthera in stigma convex (not concave) and sepals mammillary-ciliate, stigma exhibits a series of evolutionary trends in part of species, from stigma single, convex, elliptic and located near rear of spur mouth (in P. stenantha) to stigma single, suddle, and located near front of spur mouth (in P. bakeriana) and to stigma double, separate and located at front of spur mouth in the other ten species. The group in Platanthera is only confined to the area from the south fringe of the Xizang (Tibet) Plateau to the Hengduan Mountain Region. It seems that the genus has been affected by intense lift of the area, causing variation and differentiation and giving rise to the group due to the long-term natural selection. Mt. Emei in Sichuan Province is the eastern limit of distribution of the group, where there are three spcies, among which two (P. deflexilabella and P. longiglandula) are endemic to the mountains. In addition, among Risleya (1 species), Diphylax (3 species) and Diplomeris (2 species), three genera typical of distribution in the Sino-Himalayan Subregion, Risleya and Diphylax have Mt. Emei as their eastern limit. Eleven species, belonging to elements of the SinoJapanese Subregion, occur only from Japan to Western Sichuan with Mt. Emei as the western limit. Among nine species, belonging to elements of the Sino-Himalayan Subregion, six occur from the Himalayas to W. Sichuan and three of them are endemic to the Hengduan Mountain Region, with Mt. Emei as their eastern limit of distribution. There are eight endemic species and one variety of orchids in Mt. Emei, making up about 11.59% of the total endemic species in the Hengduan Mountain Region. Orchid floristic elements in Mt. Emei are obviously different from those in Mt. Jinfo, the former being mainly of the Sino-Himalayan Subregion, while the latter being mainly of the Sino-Japanese Subregion. From the distribution patterns of the orchid floristic elements in the Hengduan Mountain Region and Eastern China, the Emei Mountain is considered important for drawing a boundary line between the Sino-Japanese Subregion and the Sino-Himalayan Subregion. The discussion may be summarized as follows: the floristic features of the orchid flora in the Hengduan Mountain Region are: (1) rich in species, complex in geographical components, eminent vicarism and differentiation, and prolific in endemic species; (2) terrestrial life form is dominant one; (3) mainly consisting of temperate and subtropical East-Asian elements, es pecially, elements of Sino-Himalayan Subregion, though with some tropical elements and elem-ents of other regions.     

Intracellular mechanisms of L-selectin induced capping.

Leucocyte adhesion to endothelial cells is a tightly regulated process involving selectins, integrins and immunoglobulin-like proteins. Cell adhesion and communication are controlled by membrane dynamics like receptor capping. Capping of surface receptors is an ubiquitous mechanism but still not well understood. Employing immunofluorescence techniques, we demonstrate that L-selectin triggering results in receptor capping of the L-selectin molecules in lymphocytes. Using pharmacological inhibitors and genetic deficient cell lines we show that this process involves intracellular signalling molecules. L-Selectin capping seems to be independent on activation of p56lck-kinase, but requires the neutral sphingomyelinase, small G proteins and the cytoskeleton. Therefore, capping of L-selectin upon stimulation might play an important role in the very early phase of lymphocyte trafficking.