Spatial serial conditioning maintained with minimal temporal contingency

Two experiments used a spatial serial conditioning paradigm to assess the effectiveness of spatially informative conditioned stimuli in eliciting tracking behavior in pigeons. The experimental paradigm consisted of the simultaneous presentation of 2 key lights (CS2 and CTRL), followed by another key light (CS1), followed by food (the unconditioned stimulus or US). CS2 and CTRL were presented in 2 of 3 possible locations, randomly assigned; CS1 was always presented in the same location as CS2. CS2 was designed to signal the spatial, but not the temporal locus of CS1; CS1 signaled the temporal locus of the US. In experiment 1, differential pecking on CS2 was observed even when CS2 was present throughout the interval between consecutive presentations of CS1, but only in a minority of pigeons; prevalence of differential pecking was enhanced when CS2 duration was halved. A control condition verified that pecking on CS2 was not due to temporal proximity between CS2 and US. Experiment 2 demonstrated the reversibility of spatial conditioning between CS2 and CTRL. Asymptotic performance never involved tracking CTRL more than CS2 for any of 16 pigeons. It is inferred that pigeons learned the spatial association between CS2 and CS1, and that temporal contingency facilitated its expression as tracking behavior.     

The glycosaminoglycan attachment regions of human aggrecan

Aggrecan possesses both chondroitin sulfate (CS) and keratan sulfate (KS) chains attached to its core protein, which reside mainly in the central region of the molecule termed the glycosaminoglycan-attachment region. This region is further subdivided into the KS-rich domain and two adjacent CS-rich domains (CS1 and CS2). The CS1 domain of the human is unique in exhibiting length polymorphism due to a variable number of tandem amino acid repeats. The focus of this work was to determine how length polymorphism affects the structure of the CS1 domain and whether CS and KS chains can coexist in the different glycosaminoglycan-attachment domains. The CS1 domain possesses several amino acid repeat sequences that divide it into three subdomains. Variation in repeat number may occur in any of these domains, with the consequence that CS1 domains of the same length may possess different amino acid sequences. There was no evidence to support the presence of KS in either the CS1 or the CS2 domains nor the presence of CS in the KS-rich domain. The structure of the CS chains was shown to vary between the CS1 and CS2 domains, particularly in the adult, with variation occurring in chain length and the sulfation of the non-reducing terminal N-acetyl galactosamine residue. CS chains in the adult CS2 domain were shorter than those in the CS1 domain and possessed disulfated terminal residues in addition to monosulfated residues. There was, however, no change in the sulfation pattern of the disaccharide repeats in the CS chains from the two domains.     

Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells

Folate–chitosan (FA–CS) conjugates synthesized by coupling FA with CS render new and improved functions because the original properties of CS are maintained and the targeting ligand of FA is incorporated. In this work, FA–CS conjugates were synthesized based on chemical linking of carboxylic group of FA with amino group of CS as confirmed by Fourier transform spectroscopy (FTIR) and nuclear magnetic resonance (¹H NMR). FA–CS conjugates displayed less crystal nature when compared to CS. The FA–CS nanoparticles (NPs) were prepared by crosslinking FA–CS conjugates with sodium tripolyphosphate (STPP). Positively charged FA–CS nanoparticles were spherical in shape with a particle size of about 100 nm. Cellular uptake of CS or FA–CS nanoparticles was assayed by fluorescent microscopy using calcein as fluorescent marker in colon cancer cells (HT-29). The FA–CS nanoparticles exhibited improved uptake of HT-29 and could become a potential targeted drug delivery system for colorectal cancer.     

人CS1-Fc融合蛋白的真核表达、蛋白纯化及生物学效应鉴定

人信号淋巴细胞激活分子F7 (SLAMF7/CS1)是一种细胞表面糖蛋白,在多发性骨髓瘤细胞中高度表达。已有研究表明CS1是多发性骨髓瘤较为灵敏且特异的生物标志物。CAR-T细胞免疫疗法是治疗多发性骨髓瘤的新方法,其中CS1 CAR-T细胞免疫疗法针对复发性难治性多发性骨髓瘤有较好的疗效。为了检测CS1 CAR-T细胞上CS1CAR的表达效率和探寻CAR-T细胞免疫疗法的辅助手段,文中制备了一种CS1-Fc融合蛋白。首先利用PCR技术从已有质粒中扩增得到CS1的胞外段序列,再通过重叠延伸PCR与人IgG1-Fc段相连。将重组片段连接至pMH3真核表达载体上,经酶切鉴定和DNA测序后,将重组质粒pMH3-CS1-Fc-his转染至中国仓鼠卵巢细胞(CHO-S)。经G418加压筛选和流式细胞术鉴定,证实CS1-Fc融合蛋白在CHO-S细胞中获得了表达。利用镍柱对CS1-Fc融合蛋白进行纯化,经Western blotting鉴定,融合蛋白的分子量约为70 kDa。流式细胞术和细胞计数分析结果显示,CS1-Fc融合蛋白能有效检测CS1 CAR的表达效率,证实了CS1-Fc融合蛋白对CS1 C...     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

The effect of cyclosporine on epidermal cells. I. Cyclosporine inhibits accessory cell functions of epidermal Langerhans cells in vitro

Although the precise mechanism of action of cyclosporine (CS) is unknown, there is substantial evidence that CS preferentially acts on T cells by impairing lymphokine production. Recent studies have demonstrated that CS may also inhibit the functions of accessory cells and APC. Since topically applied CS inhibits contact sensitivity and epidermal Langerhans cells (LC) are very effective accessory cells and APC, we determined whether CS directly affects their accessory cell functions. Murine LC were pulsed with solvent control or with various doses of CS (up to 10 micrograms/ml) and then Con A-induced T cell proliferation was assayed. CS pulsing of LC caused, when compared with solvent control-pulsed LC, a dose-dependent decrease in T cell stimulation (up to 93%). LC fixed with paraformaldehyde after 2-h CS pulsing showed a similar degree of decreased accessory cell function, indicating that the immunosuppressive action is established by 2 h. The inhibitory capacity of CS pulsing on LC is not likely to be related to diminished IL-1 production, enhanced PG biosynthesis, or decreased surface Ia Ag intensity. The possibility of carryover of CS into the culture supernatants was ruled out by adding CS-pulsed LC or their supernatants to other T cell proliferative assays. Thus, these studies indicate that CS directly inhibits accessory cell functions of LC.     

Homozygous haemoglobin constant spring: A need for revision of concept

Summary Twenty-two patients with mild haemolytic anaemia and haemoglobin (Hb) Constant Spring (CS) of around 6% were studied because they were suspected of having homozygous Hb CS. Family studies revealed Hb CS trait in both parents of eight patients, supporting that they were homozygous for Hb CS. The other patients were included because they had clinical and haematological features similar to the diagnosed cases of homozygous Hb CS. Heterozygosity and homozygosity for Hb CS are clearly distinguishable in that the former is asymptomatic but the latter is associated with overt haemolytic anaemia, and the levels of Hb CS in the two conditions of less than 1% and around 6%, respectively, do not overlap. The findings in homozygous Hb CS contracdict prediction. There are four a-structural genes per normal human diploid genome. Hb CS trait is believed to be almost equivalent to a-thalassaemia 2 or a loss of one a-gene because HB CS, an a-variant, is barely or not detectable. Homozygosity for Hb CS has thus been predicted to be equivalent to a-thalassaemia 1 or a loss of two genes. The latter is asymptomatic and associated with microcytic-hypochromic red cells. However, Hb CS homozygosity presents with mild overt haemolytic anaemia and normal sized red cells. Pathogenesis associated with Hb CS inheritance is more complex than originally believed. There is a possibility that the unstable a ^CS mRNAs precipitate and aggregate leading to pathology of red cells and to the basophilic stippling appearance, so striking in this syndrome.     

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.     

Epiphyte associations and canopy soil volume: nutrient capital and factors influencing soil retention in the canopy

• Canopy soil (CS) volume reflect epiphyte community maturity, but little is known about the factors that retain CS or species succession within it. Humus fern species (e.g. Phlebodium areolatum) appear capable of retaining CS.
• In ten Quercus spp. we sampled 987 epiphyte mats to examine the role of the common epiphyte species and crown traits determining CS volume, in order to infer successional stages and identify pioneer and late successional species. Branch traits (height, diameter and slope), CS volume and cover of the epiphyte species were determined for each mat. Nutrient content was determined in CS random samples of 12 epiphyte associations and sizes (one sample from each size quintile).
• A total of 60% of the mats lack CS. Cover of P. areolatum was the main variable explaining CS volume, and this species was present in 46.8% of those with CS. Epiphyte composition was highly variable, but pioneer (species appearing in monospecific mats, without CS) and late successional species could be identified. Canopy soil nutrient content was similar among the associations of epiphytes. Magnesium, Ca and pH decreased with CS volume, while P and N increased.
• Phlebodium areolatum is associated with high CS volumes and could act as a key species in its retention. Monospecific mats of pioneer species lack CS or have low volumes, while CS is much higher in mats with late successional species, but the mechanisms of CS formation and nutrient retention in response to interactions between epiphyte species remain to be tested.

     

Serotonin derivative, N-(p-Coumaroyl)serotonin, isolated from safflower (Carthamus tinctorius L.) oil cake augments the proliferation of normal human and mouse fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF)

N-(p-Coumaroyl)serotonin (CS) with antioxidative activity is present in safflower oil. We have reported that CS inhibits proinflammatory cytokine generation from human monocytes in vitro. As reactive oxygen species (ROS) affect cell proliferation, in this study the effect of CS on the proliferation of various cell types was examined. CS augments the proliferation of normal human and mouse fibroblast cells. The cells continue to proliferate in the presence of CS and form a transformed cell-like focus without transformation. CS, however, does not augment the proliferation of other cell types, either normal or tumor cells. CS augments the proliferation of fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), but not with acidic FGF(aFGF) or platelet-derived growth factor (PDGF). This study using synthesized derivatives of CS reveals that the growth-promoting activity is not due to antioxidative activity. These findings indicate that CS is a natural compound with unique growth-promoting activity for fibroblasts.     

Dominant-negative mutant dynein allows spontaneous centrosome assembly, uncouples chromosome and centrosome cycles

Cleavage cycles commence and chromosome and centrosome cycles proceed in harmony following fertilization of Drosophila eggs and completion of the meiotic divisions. The sperm-introduced centrioles replicate, separate, and while recruit pericentriolar material centrosomes (CS) form. The CS nucleate asters of microtubules (MT). Spindles form following interaction of some astral MT with kinetochores. In unfertilized eggs, chromosomes do not replicate, and CS and MT asters never form, although their components are present in the egg cytoplasm; unknown mechanisms prevent chromosome replication and CS and MT assembly. In unfertilized Laborc(D) eggs, rudimentary CS assemble spontaneously and instantaneously and nucleate small MT asters. In fertilized Laborc(D) eggs, normal CS form and organize normal asters. However, the CS replicate prior to accomplishment of the first mitosis, and spindles with multiple CS develop. In fertilized Laborc(D) eggs, while the chromosome cycles cease, CS cycles proceed as in wild type. Knowing that Laborc(D) is a dominant-negative mutation and encodes the formation of mutant cytoplasmic dynein heavy chain molecules, we show here that cytoplasmic dynein is involved in prevention of CS assembly in unfertilized eggs and establishing harmony between the chromosome and the CS cycles.     

Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.     

Sulfatide Recognition by Colonization Factor Antigen CS6 from Enterotoxigenic Escherichia coli

The first step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections is adhesion of the bacterium to the small intestinal epithelium. Adhesion of ETEC is mediated by a number of antigenically distinct colonization factors, and among these, one of the most commonly detected is the non-fimbrial adhesin coli surface antigen 6 (CS6). The potential carbohydrate recognition by CS6 was investigated by binding of recombinant CS6-expressing E. coli and purified CS6 protein to a large number of variant glycosphingolipids separated on thin-layer chromatograms. Thereby, a highly specific binding of the CS6-expressing E. coli, and the purified CS6 protein, to sulfatide (SO₃-3Galβ1Cer) was obtained. The binding of the CS6 protein and CS6-expressing bacteria to sulfatide was inhibited by dextran sulfate, but not by dextran, heparin, galactose 4-sulfate or galactose 6-sulfate. When using recombinantly expressed and purified CssA and CssB subunits of the CS6 complex, sulfatide binding was obtained with the CssB subunit, demonstrating that the glycosphingolipid binding capacity of CS6 resides within this subunit. CS6-binding sulfatide was present in the small intestine of species susceptible to CS6-mediated infection, e.g. humans and rabbits, but lacking in species not affected by CS6 ETEC, e.g. mice. The ability of CS6-expressing ETEC to adhere to sulfatide in target small intestinal epithelium may thus contribute to virulence.