Antitumor response to recombinant murine interferon γ correlates with enhanced immune function of organ-associated,but not recirculating cytolytic T lymphocytes and macrophages

The mechanism of therapeutic activity for recombinant murine interferon-(rMu IFN) in the treatment of metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the tumor-bearing organ) were tested after 1 week and 3 weeks of rMu IFN administration (i.v. three times per week). Natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocyte (CTL) activities against specific and nonspecific targets, and macrophage tumoristatic activity were measured. rMu IFN demonstrated immunomodulatory activity in most assays of immune function. The optimal therapeutic protocol of rMu IFN (2.5×10⁶ U/kg, three times per week) prolonged survival and decreased the number of pulmonary metastatic foci. This therapeutic activity was correlated with specific CTL activity from pulmonary parenchymal mononuclear cells (PPMC), but not from spleen or blood. Macrophage tumoristatic activity in PPMC also correlated with therapeutic activity, but activity in alveolar macrophages did not. However, therapeutic activity did not correlate with NK or LAK activity at any site. These results demonstrate that the optimal therapeutic protocol is the same as the optimal immunomodulatory dose for pulmonary CTL and macrophage activities. Furthermore, while immunological monitoring may help to optimize treatment protocols, current monitoring procedures that use readily accessible sites, particularly peripheral blood, may not accurately predict the therapeutic efficacy of biological response modifiers in clinical trials.By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleThis research was sponsored by the DHHS, under contract N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

Photoreversible modulators of Escherichia coli beta-galactosidase. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

-Galactosidase (EC 3.2.1.23) is known to be inhibited by some thiol reagents. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (1) was shown to be an irreversible inhibitor, while 1, 1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2) was demonstrated as a positive irreversible modulator causing a rise of up to 186% in -galactosidase activity. Compound 2 is, however, an irreversible inhibitor of the cysteine proteinase papain (preceding paper). Kinetic values of -galactosidase at pH 8.3 with o-nitrophenyl -D-galactopyranoside (ONPG) as the substrate and for compounds 1 and 2 were determined and in view of model experiments, it was assumed that both compounds possibly reacted with the thiol side chain of Cys in the active site inducing allosteric changes in the enzyme. Since the enzyme, modified by compound 1 or 2, was a 2-nitrobenzyl derivative, near-UV irradiation resulted in a recovery of up to 91% and a reduction of the enzyme's activity to 90%, respectively.     

免疫活性细胞回输造成丙型肝炎病毒准种构成一过性改变

观察持续性感染者外周血HCV准种构成及其在增强的免疫压力下的变化规律。应用基因扩增、分子克隆和测序的方法,对4例接受自体免疫活性细胞回输的丙型肝炎患者系列血清中的HCVC区基因片段,进行了序列分析及遗传进化关系比较。HCV病毒池中均以遗传密切相关的优势准种群为主,当机体免疫状态发生变化时,4例中有3名患者血清HCV准种构成发生了一过性改变,优势准群被弱势准群替代。随后其中两例的准种构成又回复到细胞回输前的状态,另一例则形成了新的优势毒株群,第四例患者在全部随访期内准种构成没有明显规律性变化。持续性感染的HCV准种构成是处于与机体免疫压力平衡的较稳定状态,且多以一个遗传密切相关的准种群为优势群体,在受到增强的免疫压力时,这种构成的平衡被打破,HCV的准种构成变化有不同的表现形式,主导毒株的规律性变化是主要的形式之一;但随时间推移,初始状态的准种构成还可回复。     

支架蛋白GIT2通过与Smad3相互作用调节其转录活性

G蛋白偶联受体激酶相互作用蛋白2(G protein-coupled receptor kinase interacting proteins 2,GIT2)是一种信号支架蛋白,可募集多种信号通路的关键分子,参与肌动蛋白细胞骨架组装、整合素介导的细胞粘附、G蛋白偶联受体的内化及胞内信号传递等生物学过程. 采用酵母双杂交实验证明,TGF-β1信号通路的转录因子Smad3是GIT2的相互作用蛋白质,内、外源免疫共沉淀实验均证实,GIT2与Smad3存在蛋白质相互作用. 报告基因实验及免疫印迹结果表明,GIT2增加Smad3的转录活性并增强TGF-β1诱导的Smad3的磷酸化.研究还发现,Git2-/-小鼠骨髓间充质干细胞(MSC)的Smad3磷酸化受到抑制,其骨形成相关靶基因的表达水平也低于Git2+/+小鼠. 本研究表明,GIT2通过与Smad3的相互作用调节其转录活性并活化TGF-β1信号通路,可能参与调节骨髓间充质干细胞的分化.     

Insight into catalysis of a unique GTPase reaction by a combined biochemical and FTIR approach

Rap1 and Rap2 are the only small guanine nucleotide-binding proteins of the Ras superfamily that do not use glutamine for GTP hydrolysis. Moreover, Rap1GAP, which stimulates the GTPase reaction of Rap1 10(5)-fold, does not have the classical "arginine finger" like RasGAP but presumably, introduces an asparagine residue into the active site. Here, we address the requirements of this unique reaction in detail by combining various biochemical methods, such as fluorescence spectroscopy, stopped-flow and time-resolved Fourier transform infrared spectroscopy (FTIR). The fluorescence spectroscopic assay monitors primarily protein-protein interaction steps, while FTIR resolves simultaneously the elementary steps of functional groups labor-free, but it is less sensitive and needs higher concentrations. Combining both methods allows us to distinguish weather mechanistic defects caused by mutation are due to affinity or due to functionality. We show that several mutations of Asn290 block catalysis. Some of the mutants, however, still form a complex with Rap1*GDP in the presence of BeF(x) but not AlF(x), supporting the notion that fluoride complexes are indicators of the ground versus transition state. Mutational analysis also shows that Thr61 is not required for catalysis. While replacement of Thr61 of Rap1 by Leu eliminates GTPase activation by Rap1GAP, the T61A and T61Q mutants have only a minor effect on catalysis, but change the relative rates of cleavage and (P(i)(-)) release. While Rap1GAP(N290A) is completely inactive on wild-type Rap1, it can act on Rap1(T61Q), arguing that Asn290 in trans has a role in catalysis similar to that of the intrinsic Gln in Ras and Rho. Finally, since FTIR works at high, and thus mostly saturating, concentrations, it can clearly separate effects on affinity from purely catalytic modifications, showing that Arg388, conserved between RapGAPs and mutated in the homologous RheBGAP Tuberin, affects binding affinity severely but has no effect on the cleavage reaction itself.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

Spatial structure of (34–65)bacterioopsin polypeptide in SDS micelles determined from nuclear magnetic resonance data

Summary The spatial structure of a synthetic 32-residue polypeptide, an analog of the membrane-spanning segment B (residues 34–65) of bacterioopsin ofHalobacterium halobium, incorporated into perdeuterated sodium dodecyl sulfate micelles, was determined from¹H NMR data. The structure determination included the following steps: (1) local sructure analysis; (2) structure calculations using the distance geometry program DIANA; (3) systematic search for energetically allowed side-chain rotamers consistent with NOESY crosspeak volumes; (4) random generation of peptide conformations in allowed conformational space. The obtained structure has a righ-handed -helicl region from Lys⁴¹ to Leu⁶² with a kink of 27 at Pro⁵⁰. The C-cap Gly⁶³ adopts a conformation with =87±6, =43±10^o typical to a left-handed helix. The N-terminal part (residues 34–40) is exposed to the aqueous phase and lacks an ordered conformation. The secondary structure of segment B in micelles is consistent with the high-resolution electron cryomicroscopy model of bacteriorhodopsin (Henderson et al. (1990)J. Mol. Biol.,213, 899–929).     

降纤酶加灯盏花素治疗大面积脑梗死疗效观察

目的　观察降纤酶加灯盏花素治疗大面积脑梗死的疗效。　方法　随机将 85例病人分为对照组40例及治疗组 45例 ,治疗组在对照组用药的基础上加用降纤酶及灯盏花素治疗。 60天后进行疗效评价。　结果　基本治愈率是治疗组为 46.7% ,对照组为 2 5 % ,两组比较 ,治疗组疗效明显优于对照组 (P <0 .0 5 )。　结论　降纤酶加灯盏花素治疗大面积脑梗死疗效好 ,疗程短。降纤酶溶栓治疗以早期 (6h内 )应用效果好 ,但超过 6h仍然有效。     

In Vitro Differences Between Astrocytes of Control and Wobbler Mice Spinal Cord

The Wobbler mouse, a model of amyotrophic lateral sclerosis (ALS), presents motorneuron degeneration and pronounced astrogliosis in the spinal cord. We have studied factors controlling astrocyte proliferation in cultures derived from Wobbler and control mice spinal cord. Basal rate of [³H]thymidine incorporation was 15 times lower in Wobbler astrocytes. While in control cultured cells interleukin-1 (IL-1) and corticosterone (CORT) significantly increased proliferation, both agents were inactive in Wobbler astrocytes. The lack of response to CORT was not due to the absence of glucocorticoid receptors, because similar receptor amounts were found in Wobbler and control astrocytes. In contrast to IL-1 and CORT, transforming growth factor-₁ (TGF-₁) substantially increased proliferation of Wobbler astrocytes but not of control cells. Differences in response to TGF-₁ were also obtained by measuring glial fibrillary acidic protein (GFAP) immunoreaction intensity, which was substantially higher in Wobbler astrocytes. Thus, abnormal responses to different mitogens characterized Wobbler astrocytes in culture. We suggest that TGF-₁ may play a role in the reactive gliosis and GFAP hyperexpression found in the degenerating spinal cord of this model of ALS.