Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans- Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.     

Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...     

Adaptation to Delayed Force Perturbations in Reaching Movements

Adaptation to deterministic force perturbations during reaching movements was extensively studied in the last few decades. Here, we use this methodology to explore the ability of the brain to adapt to a delayed velocity-dependent force field. Two groups of subjects preformed a standard reaching experiment under a velocity dependent force field. The force was either immediately proportional to the current velocity (Control) or lagged it by 50 ms (Test). The results demonstrate clear adaptation to the delayed force perturbations. Deviations from a straight line during catch trials were shifted in time compared to post-adaptation to a non-delayed velocity dependent field (Control), indicating expectation to the delayed force field. Adaptation to force fields is considered to be a process in which the motor system predicts the forces to be expected based on the state that a limb will assume in response to motor commands. This study demonstrates for the first time that the temporal window of this prediction needs not to be fixed. This is relevant to the ability of the adaptive mechanisms to compensate for variability in the transmission of information across the sensory-motor system.     

Interspecific variability in sensitivity to UV radiation and subsequent recovery in selected isolates of marine bacteria

The interspecific variability in the sensitivity of marine bacterial isolates to UV-B (295- to 320-nm) radiation and their ability to recover from previous UV-B stress were examined. Isolates originating from different microenvironments of the northern Adriatic Sea were transferred to aged seawater and exposed to artificial UV-B radiation for 4 h and subsequently to different radiation regimens excluding UV-B to determine the recovery from UV-B stress. Bacterial activity was assessed by thymidine and leucine incorporation measurements prior to and immediately after the exposure to UV-B and after the subsequent exposure to the different radiation regimens. Large interspecific differences among the 11 bacterial isolates were found in the sensitivity to UV-B, ranging from 21 to 92% inhibition of leucine incorporation compared to the bacterial activity measured in dark controls and from 14 to 84% for thymidine incorporation. Interspecific differences in the recovery from the UV stress were also large. An inverse relation was detectable between the ability to recover under dark conditions and the recovery under photosynthetic active radiation (400 to 700 nm). The observed large interspecific differences in the sensitivity to UV-B radiation and even more so in the subsequent recovery from UV-B stress are not related to the prevailing radiation conditions of the microhabitats from which the bacterial isolates originate. Based on our investigations on the 11 marine isolates, we conclude that there are large interspecific differences in the sensitivity to UV-B radiation and even larger differences in the mechanisms of recovery from previous UV stress. This might lead to UV-mediated shifts in the bacterioplankton community composition in marine surface waters.     

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.     

Transmitter-Induced Calcium Responses Differ in Astrocytes Acutely Isolated from Rat Brain and in Culture

Abstract: Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3–10-day-old rats frequently showed increased intracellular Ca²⁺ concentration responses to l -glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca²⁺ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of, or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca²⁺ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.     

The I/LWEQ Domain in RapGAP3 Required for Posterior Localization in Migrating Cells

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap-GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap-GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.     

Sporophytic response to pollen selection for Alachlor tolerance in maize

In order to assess the efficiency of male gametophytic selection (MGS) for crop improvement, pollen selection for tolerance to herbicide was applied in maize. The experiment was designed to test the parallel reactivity to Alachlor of pollen and plants grown in controlled conditions or in the field, the response to pollen selection in the sporophytic progeny, the response to a second cycle of MGS, and the transmission of the selected trait to the following generations. The results demonstrated that pollen assay can be used to predict Alachlor tolerance under field conditions and to monitor the response to selection. A positive response to selection applied to pollen in the sporophytic progeny was obtained in diverse genetic backgrounds, indicating that the technique can be generally included in standard breeding programs; the analysis of the data produced in a second selection cycle indicated that the selected trait is maintained in the next generation.     

The safe face lift with bony anatomic landmarks to elevate the SMAS

The risk for facial nerve injury has been reported to be increased with the inclusion of superficial musculoaponeurotic system (SMAS) elevation as compared with a skin-only face lift. The facial nerve courses through the parotid gland. The SMAS is elevated superficial to the parotid gland. However, in elevating the SMAS anterior to the parotid gland, the facial nerve is at risk of injury where its branches emerge from the anterior edge of the parotid gland. The purpose of this study was to identify bony anatomic landmarks to predict the location of the anterior edge of the parotid gland to avoid injury to the facial nerve branches as they exit the parotid gland. The authors dissected 20 cadaver face halves to determine bony landmarks-the masseteric tuberosity and the inferior lateral orbital rim-to predict the location of the anterior parotid edge. Then they measured the anterior edge of the parotid gland in relation to the vector formed between these two bony landmarks. They identified and measured the most anterior portion of the parotid gland in relation to this vector. Then the most posterior aspect of the parotid gland in relation to this vector was measured. In the 20 dissections, the authors found the most anterior portion of the parotid gland to be 2.7 +/- 1.0 mm anterior to the vector from the inferior lateral orbital rim to the masseteric tuberosity. The most posterior part of the anterior edge of the parotid gland in relation to this vector was found to be 1.0 +/- 1.5 mm posterior to this vector. The parotid gland measured an average of 38.8 +/- 3.5 mm in width from the tragus to the anterior parotid edge. In elevating the SMAS with a face lift, the facial nerve branches can be predicted to exit the anterior edge of the parotid gland, which can be located 38.8 mm anterior to the tragus and near the vector from the inferior lateral orbital wall to the masseteric tuberosity.     

Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation

To analyze the regulation of PRL gene expression by thyroid hormone (T3), fusion gene constructs containing various lengths of the rat PRL gene 5'-flanking sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5'-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T3. A construct containing the proximal enhancer region (positions -292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T3 with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T3 with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer region revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T3. The stimulatory response to T3 was additive with the response to estradiol, suggesting distinct elements, but deletion to position -1565 abolished the response to estradiol and permitted an inhibitory response to T3. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T3. Mutation of the sequence GGTCA at positions -1555 to -1551 resulted in an inhibitory response to T3, implicating this sequence in the stimulatory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

激光采血的分析研究

本文根据激光与皮肤组织的作用规律对紫外激光,可见激光和红外激光的采血效果进行了系统地分析研究,研究结果表明：CO2激光与Er:YAG激光是采血的理想工具。并对红外激光采血的参娄进行了定量地分析与计算,为激光采血设备的研制和应用提供一个参考。     

儿童毒蛇咬伤的特点与程序化救治的疗效观察

目的通过分析儿童毒蛇咬伤的特点,评价程序化综合救治方法对儿童毒蛇咬伤的疗效。方法通过观察17例毒蛇咬伤儿童的临床特点,尽早进行病情预测,同时根据毒蛇咬伤的临床病情分型予程序化综合救治。监测病情变化、伤口愈合情况及平均住院时间。结果儿童毒蛇咬伤的特点为不明种类毒蛇咬伤多,症状较成人严重,易发生并发症。经过早期程序化综合救治总有效率100%,伤口渗血、肿胀明显缓解,无1例死亡,平均留观时间4.5天。结论根据儿童毒蛇咬伤特点,早期准确预测和尽快应用程序化救治能提高毒蛇咬伤疗效。     

Specificity of the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase and to a series of other calcium-binding proteins

Trifluoperazine inhibits the activation of phosphodiesterase by binding to the calcium-dependent activator. To determine further the specificity by which trifluoperazine binds to activator, we compared the binding of trifluoperazine to activator prepared from several species and tissues and to a number of other calcium-binding proteins devoid of activator activity.Trifluoperazine binds to activator prepared from human, bovine, rat and rabbit brain and from chick embryo fibroblasts. In each case, the binding of trifluoperazine to activator was qualitatively similar and related quantitatively to the ability of the preparation to activate phosphodiesterase.Of the other calcium-binding proteins examined, namely, troponin-C, S-100 protein, phospholipase A, phospholipase B and myosin light chain, only troponin-C displayed any significant calcium-specific binding of trifluoperazine. The binding to troponin-C, however, appeared to be different from the binding to activator; whereas the binding of trifluoperazine to actovator showed no cooperativity, the binding to troponin-C showed positive cooperatively.These results and earlier data showing that trifluoperazine fails to bind to a variety of other proteins, indicate that the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase is selective and suggest that this binding may explain some of the biochemical and pharmacological actions of this antipsychotic agent.     

In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster

Summary Pinocytotic activity has been analyzed in Drosophila oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm.In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone.     

Individuation of monoclonal anti-HPV16 E7 antibody linear peptide epitope by computational biology

We applied computational biology to identify the linear amino acid sequence recognized by a mouse monoclonal antibody raised against the full length HPV16 E7 oncoprotein. Computer-assisted search for the epitopic peptide used two parameters: the capability of E7 peptides to bind to MHC class II molecules, and the similarity level of the oncoprotein sequence to the mouse proteome. We report that anti-E7 mAb recognized the peptide having both high binding potential to MHC II molecules and low level of molecular mimicry to mouse proteome. Peptide ability to bind to MHC II molecules appears a necessary but not sufficient condition to determine peptide immunodominance, by needing to be supported by a low degree of peptide similarity to the host’s proteome.     

Observations on the effects of vibration and noise on plasma ACTH and zinc levels, pregnancy and respiration rate in the guineapig

The responses of female guineapigs to vibration and noise were examined with the use of an apparatus designed to simulate transport. Peak levels of plasma ACTH and zinc concentrations were attained after 4 min of exposure to vibration and 80-90 dB of noise. The respiration rate, normally around 90/min, was increased to 103/min when the animals were moved (while still in their cages) to the experimental area; it rose to 129/min when the apparatus was switched on to expose the animals to vibration and noise. Those left adjacent to the apparatus and exposed to noise alone elevated their respiration rates to 115/min. Respiration rates returned to normal within 2.5 h. There was no apparent effect on the maintenance of pregnancy, gestation length, litter size or post-partum growth of the young born to guineapigs exposed to this vibration and noise for a period of 1 h at mid-term.     

Pollenkitt – its composition, forms and functions

Two types of sticky pollen coat material exist in angiosperms, both produced by the anther tapetum. Pollenkitt is the most common adhesive material present around pollen grains of almost all angiosperms pollinated by animals, whereas tryphine seems to be restricted only to Brassicaceae. Tapetal cell protoplasts have different patterns of development according to the products formed during their development and degeneration. If tryphine is formed, the tapetal cell protoplasts lose their individuality at the microspore stage. If pollenkitt is formed, their contents degenerate at later stages. Cell content is totally reabsorbed, when ripe pollen is not surrounded by any gluing material. Current knowledge of pollenkitt formation, deposition on pollen grains and chemical composition are reviewed and discussed. Methods for detecting this viscous fluid are also presented. The many functions of pollenkitt, deduced from personal observations and the literature, act in the period between anther opening and pollen hydration on the stigma; they are: (1) to hold pollen in the anther until dispersal; (2) to enable secondary pollen presentation; (3) to facilitate pollen dispersal; (4) to protect pollen from water loss; (5) to protect pollen from ultra-violet radiation; (6) to maintain sporophytic proteins responsible for pollen–stigma recognition inside exine cavities; (7) to protect pollen protoplasts from fungi and bacteria; (8) to keep together pollen grains during transport; (9) to protect pollen from hydrolysis and exocellular enzymes; (10) to render pollen attractive to animals; (11) to render pollen visible to animal eyes; (12) to hide pollen from animal eyes; (13) to avoid predation of pollen through smell; (14) to enable adhesion to insect bodies; (15) to enable pollen packaging by bees and to form corbicules; (16) to provide a digestible reward for pollinators; (17) to enable pollen clumps to reach the stigma; (18) to allow self-pollination; (19) to facilitate adhesion to the stigma; (20) to facilitate pollen rehydration. Depending on the developmental program of the species, these functions may act during pollen presentation, in relation to pollinators, during pollen dispersal and when pollen reaches the stigma.     

西双版纳热带雨林蚁科昆虫区系分析

在西双版纳热带雨林已鉴定蚊科昆虫９亚科７６属２６７种。西双版纳地区的蚂蚁区系以热带至亚热带分布的东洋界成分最为丰富。在属级水平上,与马来西亚界关系最为密切。与澳洲界关系较密切;与非洲界和马拉加西界的关系知中。与新北界,新热带界和古北界的关系最为疏远。可见西双版纳的蚂蚁区系具有典型的热带亚洲起源特征,同时与澳洲和非洲的热带区系有一定的渊源关系。