Cell surface proteins in the early embryogenesis of Pleurodeles waltlii

Surface proteins in the first embryonic stages (8–32 cells, morula, blastula, early and late gastrula) of Pleurodeles waltlii were selectively labelled by ¹²⁵I using lactoperoxidase and glucose/glucose oxidase. Iodination was effected either on non-dissociated embryos or after their dissociation with EDTA. On the outer surface of non-dissociated embryos the two-dimensional electrophoresis revealed only three groups of ¹²⁵I-labelled proteins which did not change during all studied stages. Quite different results were obtained with the cells of dissociated embryos. In addition to the iodinated proteins of the embryonic outer surface seven major iodinated proteins were identified. These proteins originate from the regions of cell-cell contacts in intact embryo. Their two-dimensional pattern in dissociated cells changes between stages 8–32 cells and morula. The next important difference was observed during gastrulation, which corresponds in Pleurodeles waltlii to the first morphogenetic movements. Therefore the outside and inside cell surfaces of embryo are different already at stage 8–32 cells (and probably earlier), before the first step of morphogenesis. The changes of cell surface proteins at early embryonal development take place inside the embryo, in the regions of cell-cell interactions.     

雌性畸胎瘤细胞离体分化时X染色体的行为

用雌性畸胎瘤LT细胞(具有两个X染色体)为材料,在离体条件下诱导分化。通过对X连锁的HPRT和G6PD等酶的定量分析,并与Pcc3/A/1畸胎瘤细胞(XO型)对比。结果表明,HPRT与G6PD酶比活性在分化后的LT细胞中,以及在已分化的胚胎体重新种植并传代后的细胞中,均与Pcc3/A/1(XO型)细胞相似,比未分化的细胞降低了一半左右。这些结果可认为,在雌性畸胎瘤细胞离体分化过程中,发生了X染色体的生化分化。     

In vitro and in vivo iodination of human thyroglobulin in relation to hormone release

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.     

Overexpressions of cDNAs for β-Amyloid Precursor Proteins 695, 751, and 770 Enhance the Secretion of β-Amyloid Precursor Protein Derivatives and the Survival of P19-Derived Neurons

Abstract: P19 is a C3H mouse-derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of β-amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19-derived neurons had a limited α-secretase activity, all of these clones produced significant amounts of secreted β-amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19-derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to β-amyloid precursor protein. These results suggest neurotrophic effects of secreted β-amyloid precursor protein and contrast with a previous report in which overexpression of a full-length cDNA for β-amyloid precursor protein led to degeneration of P19-derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C-terminal fragments of β-amyloid precursor protein; moreover, P19-derived neurons differ from P19 stem cells in the processing of these C-terminal fragments.     

Tyrosine residues modification studied by MALDI-TOF mass spectrometry

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.     

Purification and characterization of a collagenous protein secreted by a murine teratocarcinoma-derived cell line

A collagenous protein could be precipitated by (NH₄)₂SO₄ from the culture medium of a murine teratocarcinoma-derived cell line (Ko, C.Y., Johnson, L.D. and Priest, R.E. (1979) Biochim. Biophys. Acta 581, 252–259). Further purification of this protein was achieved by combining DEAE-cellulose chromatography with either CM-cellulose or molecular sieve chromatography. The collagenous polypeptides had subunit molecular weights of 160 000, if determined by molecular sieve chromatography, or 190 000, if determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they are not linked by disulphide bridges. Amino acid composition of this collagen is similar to that of a murine type IV collagen isolated from EHS sarcoma (Timpl et al. (1978) Eur. J. Biochem. 84, 43–52). The most prominent peptides resulting from cleavage of the protein by CNBr had estimated molecular weights of 25 000, 23 000, 11 700 and 9400. Pepsin treatment of this collagen under non-denaturing conditions produced three major fragments having molecular weights of 70 000, 45 000 and 43 000. We conclude that the collagen secreted by the murine teratocarcinoma-derived cell culture is a type IV basement membrane collagen. Therefore, this culture system should provide a continuous source of type IV collagen, which may be used to study the interaction of this collagen with other basement membrane components.     

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis

Glutamate-induced elevation in intracellular Ca²⁺ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca²⁺-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca²⁺ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca²⁺ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes.     

BMP2/7异源二聚体调控CIZ的表达与自身活性的关系

BMP2/7异源二聚体的活性显著高于BMP2同源二聚体,但其机制并不清楚。采用哺乳动物细胞表达的BMP2/7异源二聚体处理成骨细胞MC3T3-E1,细胞化学染色发现BMP2/7的活性显著高于BMP2,报告载体p3GC2-LUX检测发现BMP2/7能够明显上调BMP/Smad通路的活性(P<0.05)。但在成骨细胞中过表CIZ（Casinteracting zinc finger protein）,能够显著抑制BMP2/7上调ALP与Osteocalcin的作用,并阻断BMP2/7对BMP/Smad通路的激活。同时发现BMP蛋白能够上调CIZ的表达,但BMP2/7的作用明显低于BMP2同源二聚体。可以认为BMP2/7能够诱导CIZ的表达,但由于作用较弱,所以对自身活性的反馈抑制作用也较弱,这可能是BMP2/7有着较强生物活性的关键所在。     

Effects of Temperature on Promastigotes of Several Species of Leishmania

ABSTRACT. Six Leishmania species were studied comparatively, in order to determine the influence of temperature "in vitro" on differentiation, infectivily and protein synthesis. Differentiation ocurred in a heterogeneous manner, even in species that produce similar clinical manifestations. Thus, no association could be found between thermosensitivity and disease. The association between expression of proteins and increasing temperatures was analyzed at 34° C by polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE), using different incubation times, and employing a technique involving metabolic incorporation of [³⁵S]-methionine. Protein synthesis was very similar in all the New World species apart from L. amazonensis , which expressed a protein of approximately 80 kDa when incubated at 34° C for 2 hours. All the tested species had in common the expression of a 70 kDa protein. Differences, however, were observed in relation to the time interval for protein expression. in L. chagasi , synthesis was detected after 30 minutes of incubation at 34° C, while L. braziliensis required 1 hour at the same temperature. The "in vivo" and "in vitro" infectivity of the differentiated forms was also analyzed, but no significant differences were observed.     

Aluminum Inhibits Calpain-Mediated Proteolysis and Induces Human Neurofilament Proteins to Form ProteaseResistant High Molecular Weight Complexes

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.     

Receptor-binding kinetics of A-14 and A-19 125I-labelled insulin

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 ¹²⁵I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.     

The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.     

Complete conformational stability of kinetically stable dimeric serine protease milin against pH,temperature, urea,and proteolysis

Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T _m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.     

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.     

The chlamydial protease CPAF: important or not,important for what?

The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.