In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.     

16种茯苓菌株液体发酵产胞内多糖与三萜的比较研究

筛选茯苓高产胞内多糖和胞内三萜的优良液体发酵出发菌株。采用PDA富集固体平板培养与液体发酵培养测定菌丝体生长速率;采用液体发酵策略分析16种茯苓菌株产胞内多糖与胞内三萜的潜能。实验结果表明菌株生长于固体培养基与种子培养基的生长速率之间没有关联性;降低一级种子培养基初始pH值到4.0时能有效缓解茯苓菌株培养物褐化现象;AS5.137胞内多糖含量最高,达377.60±0.10 mg/g,而DB菌株显示出最高的胞内多糖产量,达1.01±0.13 g/L;Y1菌株胞内三萜含量最高,达83.89±4.28 mg/g,而Jingzhou28菌株胞内三萜产量最高,达136.63±26.66 mg/L。就生产茯苓胞内多糖与胞内三萜而言,AS5.137与DB菌株适合作为液体发酵产胞内多糖的出发菌株;Y1,Jingzhou28,Z(z)与Xingpinzhong菌株均较适合作为液体发酵产胞内三萜的出发菌株。     

Effector proteins encoded by Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton after translocation into host cells

The facultative intracellular pathogen Salmonella enterica has evolved strategies to modify its fate inside host cells. One key virulence factor for the intracellular pathogenesis is the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2). We have previously described SPI2-encoded SseF and SseG as effector proteins that are translocated by intracellular Salmonella . Detailed analysis of the subcellular localization of SseF and SseG within the host cell indicated that these effector proteins are associated with endosomal membranes as well as with microtubules. Specific association with microtubules was observed after translocation by intracellular Salmonella as well as after expression by transfection vectors. In epithelial cells infected with Salmonella , both SseF and SseG are required for the aggregation of endosomal compartments along microtubules and to induce the formation of massive bundles of microtubules. These observations demonstrate that SPI2 effectors interfere with the microtubule cytoskeleton and suggest that microtubule-dependent host cell functions such as vesicle transport or organelle positioning are altered by intracellular Salmonella .     

Glutamine synthetase in alder (Alnus glutinosa) root nodules. Purification, properties and cytoimmunochemical localization

Chromatographic, kinetic and regulatory properties of glutamine synthetase (GS) were investigated in root nodules of Alnus glutinosa L. (Gaertn) grown in a culture chamber. By DEAE Sephacel column chromatography and polyacrylamide gel electrophoresis, a major form of the enzyme was identified. Molecular weight was about 360 000 for the native protein and 43 000 for the subunits. Optimum pH was about 7.3 and Km for glutamate and ATP were 0.9 m M and 0.5 m M , respectively. By immunofluorescent techniques GS was localized in the inner cortical cells but not in vesicles of alder root nodules.     

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na,Ca, and H

Summary The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basallateral Na pump with ouabain. With high Na activity in the mucosal medium (86mm), increases in intracellular Na activity from 10 to over 40mm increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1mm had no effect on the change inP _Na with increasing Na_c, but increasing serosal Ca to 5mm enhanced the reduction inP _Na with increasing Na _c , presumably by increasing Ca influx into the cell.P _Na was also reduced by serosal vanadate (0.5mm), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO₂, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloridesensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of theI-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions onP _Na are voltage independent.     

SMAD蛋白家族:一类新的细胞内信号传导蛋白

SMADs是新近发观的一族细胞内信号传导蛋白,包括8个成员,即SMAD1～8。SMAD1、2、3、5和8是一类,它们被TGF-β受体或BMP受体激活而磷酸化,称为受体调节SMAD,传导TGF-β或BMP的信号。SMAD6和7是另一类,它们抑制受体调节SMAD传导信号。SMAD4是第2类,它是受体调节SMAD传导信号的伴侣。受体调节SMAD传导信号必须先与SMAD4结合形成异源复合物,才能进到核中,调节转录活动。本文简要介绍了各成员的特性及作用。     

Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 µg/ml. The highest Ca²⁺ rise (104 ± 47 nM) was observed for 10 µg/ml SSP. It was mainly dependent (81 ± 11%) on extracellular calcium influx, however, SSP mobilized 68 ± 7% of Ca²⁺ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca²⁺ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca² concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.     

Anaplasma phagocytophilum Rab10‐dependent parasitism of the trans‐Golgi network is critical for completion of the infection cycle

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell‐derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense‐cored (DC) morphotypes. Host–pathogen interactions that are critical for RC‐to‐DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)‐tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid‐rich trans‐Golgi network (TGN) to its vacuole in a guanine nucleotide‐independent manner. Here, we demonstrate that endogenous Rab10‐positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum‐occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up‐regulates expression of the DC‐specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide‐independent, Rab10‐specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens.     

Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration

Summary Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pH_i or [Ca²⁺]_i in crayfish septate axons uncoupled by acidification. For measuring [Ca²⁺]_i a new neutral carrier sensor sensitive to picomolar [Ca²⁺] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pH_i or [H⁺]_i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pH_i and junctional resistance curves caused significant hysteresis in the pH_i versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca²⁺]_i, increased by approximately one order of magnitude from basal values of 0.1–0.3 m. The curves describing the time course of changes in [Ca²⁺]_i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca²⁺]_i recovered following a single exponential decay with a time constant of 2 min. Different rates of acidification caused increases in [Ca²⁺]_i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca²⁺]_i than to [H⁺]_i.     

The intracellular metabolism of iodine in carcinogenesis

Research from this laboratory and others have concluded that significant glandular atypia, and often neoplasia, occurs in the breast tissues of rodents and humans under conditions of iodine deprivation. These cellular changes caused by iodine deficiency are intensified, by aging, steroid hormones, and pituitary hormones. There has been controversy concerning the effect of iodine deficiency on stimulation and maintenance of cancer of the breast in rodents when the cancer is induced chemically or by transplantation. However, neither within this induced neoplastic framework nor with the dysplastic changes seen by deficiency alone have laboratory studies of thepathway of intracellular iodine been previously possible. The new research data addresses the question of whether organification occurs and whether iodine significantly affects the intracellular structures. An hypothesis will be presented that places the inorganic element, iodine, into association with receptor protein complexes that may be responsible for intracellular sex hormone activity. The relationship of this mechanism to carcinogenesis in breast tissue will be considered.     

Is the intrasomal phase of fast axonal transport driven by oscillations of intracellular calcium?

An hypothesis is presented suggesting that the delivery of vesicle-packaged protein from the neuronal soma to the axonal transport system is physiologically coupled to spontaneous fluctuations of intracellular calcium (Ca_i). Evidence is reviewed that oscillations of Ca_i, commonly detected as agonist-or voltage-triggered waves and spikes propagating through the cytosol, also occur as spontaneous events. Endogenously-generated oscillations are examined since intrasomal transport persists in the absence of extracellular signals or nerve impulse activity. Vesicle budding from the endoplasmic reticulum (ER) may be a key step at which anterograde transport is regulated by events related to the release and reuptake of ER stores of Ca²⁺.Special-issue dedicated to Dr. Sidney Ochs.     

“Calcium receptors” on eukaryotic cells with special reference to the osteoclast

There is a growing list of cells that are capable of detecting and responding to changes in the concentration of extracellular calcium. The two classic examples of this behaviour are the calcitonin-secreting parafollicular cells of the thyroid and parathyroid hormone-secreting chief cells of the parathyroid gland. A more recent addition to this list is the renin-secreting juxtaglomerular cell of the kidney. Particularly intriguing has been independently the discovery by two laboratories, that the resorptive cell of bone, the osteoclast, is capable of detecting changes in ambient calcium. A common theme amongst all these so called calcium-responsive cells is that extracellular calcium increases elevate intracellular calcium levels, and this intracellular signal is either stimulatory or inhibitory to the functional response. But how these cells detect changes in the concentration of extracellular calcium, and how these recognition events are subsequently transformed into intracellular signals that regulate cell function are somewhat less clear. The commentary reveals some recent developments that seemingly provide insights into these mechanisms, with special reference to the osteoclast.     

Synaptic mechanisms of monaural and binaural processing in the locust

(1) Responses of auditory interneurones were recorded intracellularly within the metathoracic ganglion of the locust when stimulating each tympanic membrane with a piezoelectric transducer. Thus, in contrast to conventional sound stimulation, each of the two ears could be activated independently from the other at variable intensities, duration and stimulus onsets. By means of this ‘earphone-like’ stimulation technique the binaural integration properties of auditory interneurons could be analysed. (2) A minority of units (3 out of 43) was affected by input from one side only. Their synaptic input was purely excitatory and the intensity characteristics reflected those of auditory receptor fibres. (3) Most interneurones received input from both ears, each being excitatory or one excitatory or one excitatory and one inhibitory. In some units the unilateral synaptic response already included both an EPSP and an IPSP. As a result of varying temporal interactions between the EPSP and the IPSP within the unilaterally evoked complex response the intensity characteristics differed widely from unit to unit. (4) With binaural simultaneous stimulation the complexity of the postsynaptic responses of most interneurones increased as the synaptic input from both ears coincided at the level of the recorded interneurone. Although both ears were stimulated symmetrically (at the same time and intensity), units were recorded where the latencies of ipsilateral and contralateral synaptic input were different. Contralateral inhibition could either follow or precede ipsilateral excitation and in some cases both EPSP and IPSP had the same latency. On the basis of these findings the binaural synaptic mechanisms of directional coding are discussed and compared with corresponding results under free field stimulus conditions.     

测定细胞内自由钙的显微荧光光度计的研制和应用

以ＸＳＪ－２型荧光显微镜为基础,建立了测定细胞内自由钙的显微荧光光度计,为建立这套装置,对荧光显微镜主要作了如下改动：首先,为提供稳定的激光光源,交流汞灯被换成直流氙灯;其次光路中安装了镶有两块干涉滤色片的激发滤色片转盘,使生物样品交地被两种不同波长的光激发。整套装置的运转在微机控制下,在这套装置上,成功地用Ｆｕｒａ－２测定了细胞内自由钙变化,文中提供了应用实例。     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

通过膜片钳玻璃微电极内插管进行胞内透析

本文介绍一种膜片钳微电极内插管进行胞内透析的方法。利用通用的微电极夹持器在其抽吸负压的侧管上方钻一斜孔直通夹持器中央管腔。插入微量移液管头拉制成的细管（外径约0.1mm）,后者与Ag-AgCl电极一起伸出夹持器口端。通过相连的注射器,可以很方便地进行电极内液置换及胞内药物透析,此法和二次钳压技术及国外介绍的微插管电极内液置换相比,更加简便易行,结果更可靠。     

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method

A method for the measurement of the cytosolic Na⁺ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na⁺ for H⁺ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H⁺ across the membrane (no membrane potential and pH_i = pH_o), addition of ionophore would induce a H⁺ flux only if there is a concentration difference for Na⁺. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na⁺] equals intrasynaptosomal [Na⁺. The intrasynaptosomal [Na⁺] concentration was determined to be 7 ± 3 mM (n = 5; mean ± S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.