不可切除胃癌转化治疗的研究进展

胃癌在全国范围内是发病率最高的恶性肿瘤之一，在癌症相关死亡原因中排名第三。目前外科手术仍然是胃癌获得根治性治疗的主要手段，但是其中有30%~40%的进展期胃癌患者在初诊时就失去了根治手术的机会，因此不可切除胃癌转化为可切除胃癌是患者获得"治愈"的最佳选择。随着近些年来新型药物的出现、治疗手段的多样化及多学科诊治模式的发展，"转化治疗"的理念应运而生，从而延长患者生存时间并提高其生活质量。本文就不可切除胃癌的治疗现状与进展、"转化治疗"的应用等进行综述。     

Terminal Differentiation of Mouse Preadipocyte Cells: The Mitogenic-Adipogenic Role of Growth Hormone is Mediated by the Protein Kinase C Signalling Pathway

Abstract

The role of growth hormone (GH) in the differentiation process of Obi 771 mouse preadipocyte cells has been studied under culture conditions that were serum-free and hormone-supplemented and which were previously shown to lead to terminal differentiation. In the absence of GH, a dramatic decrease in the adipogenic activity of the culture medium could be observed, as indicated 12 days after confluence by the low levels of glycerol-3-phosphate dehydrogenase activity and the sharp reduction of the number of triacylglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel loss of the mitogenic potency of the culture medium. Determination of the half-maximal and maximal concentrations of GH required for the restoration of growth and differentiation were identical, 0.5 and 2 nM, respectively. Despite the presence of insulin-like growth factor-I (IGF-I) to substitute for supraphysiological concentrations of insulin and to saturate IGF-I receptor, GH was still required to induce terminal differentiation of a maximal number of cells. However, protein kinase C activators such as prostaglandin F₂a, phorbol esters and diacylglycerol were able to mimic GH in promoting a maximal mitogenic-adipogenic response, indicating that the ability of GH to induce diacylglycerol production (Doglio et al., 1989; Catalioto et al., 1990) plays a prominent role in this process. Furthermore, in agreement with the fact that the mitoses which precede terminal differentiation of Obi 771 preadipocytes are strictly controlled by cAMP and only modulated by protein kinase C., terminal differentiation of Ob 1771 preadipocytes occured in the absence of GH upon supplementation with high concentrations of carbaprostacyclin, added as a cAMP-elevating agent or with 8-Br-cAMP, added as a cAMP analogue. It is concluded that the control exerted by GH on terminal differentiation of mouse preadipocytes corresponds to a modulating mitogenic effect mediated through protein kinase C activation and leading to a potentiation of the cAMP and IGF-I mitogenic signalling pathways.     

Clinical phenotypes of spinal muscular atrophy patients with hybrid SMN gene

BackgroundSpinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype.MethodWe analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR.ResultsSMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene.ConclusionHybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.     

Improvement on properties of experimental resin cements containing an iodonium salt cured under challenging polymerization conditions

ObjectiveThe use of resin cements in clinical practice entails photopolymerization through prosthetic devices, which precludes light penetration. The objective of this study was to modify experimental resin cements (ERCs) with diphenyliodonium hexafluorophosphate (DPI) in an attempt to improve chemical and mechanical properties of materials cured with reduced irradiance and final radiant exposure.MethodsA co-monomer base containing a 1:1 mass ratio of 2.2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bis-GMA) and triethyleneglycol dimethacrylate (TEGDMA) was prepared, with 1 mol% of camphorquinone and 2 mol% of ethyl 4-(dimethylamino)benzoate as initiator system. The resin was divided into 4 fractions according to the DPI concentrations (0, 0.5, 1 and 2 mol%). The challenging polymerization condition was simulated performing the light activation (12, 23 and 46 s) through a ceramic block (3 mm thick). The irradiance was assessed with a calibrated spectrometer (1320 mW/cm²), resulting in three levels of radiant exposure (0.58, 1.1 and 2.2 J/cm²). The polymerization kinetics was evaluated in real-time using a spectrometer (Near-IR). Water sorption and solubility was analyzed and the cohesive strength of resins obtained through the microtensile test. Polymerization stress was assessed by Bioman method.ResultsResins containing DPI had higher degree of conversion and rate of polymerization than the control (without DPI). The use of DPI reduced water sorption and solubility, and led to higher cohesive strength compared to resins without the iodonium salt. However, the stress of polymerization was higher for experimental resins with DPI.SignificanceEven under remarkably reduced irradiance, cements containing a ternary initiating system with an iodonium salt can present an optimal degree of conversion and chemical/mechanical properties.     

Converting diameter measurements of Pinus radiata taken at different breast heights

Two compatible conversion factors for converting diameter measurements taken at different breast heights were derived for Pinus radiata using taper data from more than 3000 trees. The two breast heights used for conversion were 1.3 and 1.4 m above ground, as defined in Australia and New Zealand, two major radiata-growing countries in the world. The conversion factors were estimated through three alternative statistical methods including simple least squares regression, seemingly unrelated regression and errors-in-variables models. The three sets of estimates were almost identical and had similar conversion accuracy, although the second method was slightly better. The conversion factors were more accurate than overbark taper equations used for the same purpose. The factor was 0.9916 for converting diameter measured at 1.3 to that at 1.4 m above ground, and the inverse of this value, 1.0084, was for the vice versa. When calculating tree and stand volume and biomass using equations with diameter at a different breast height as a predictor to that of the input data, the bias, either over or under estimation, could be between 1.67% and 2.00% without conversion. These conversion factors will facilitate the sharing of data among radiata growing countries with different definitions of breast height, but more importantly it will help correct the bias in stand volume and biomass estimation caused by the seemingly negligible difference in breast height when software for forest resource management and decision support developed in one country is applied in another. Such bias when accumulated over a large management area may not be financially insignificant for an astute forest management agency.     

Polarized gene conversion at the bz locus of maize

Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.Gene conversion in organisms where all four products of a meiotic tetrad can be recovered refers to a departure from the normal 2:2 segregation of alleles and arises from the repair of meiotic double-strand breaks (DSBs) by a homologous recombination mechanism (1). Gene conversion represents the nonreciprocal, but faithful, transfer of information between two homologous DNA sequences, usually located in homologous chromosomes. The stretch of DNA that is transferred during a gene conversion event, called the conversion tract, can vary in yeast from a few hundred bases to more than 12 kb and is composed of sequences found in only one of the parental chromosomes, i.e., it is continuous, not patchy. Conversion polarity within a gene, which is the tendency of markers located near one end of the gene to convert more frequently than those located at the opposite end, has been reported in several Ascomycete fungi: Ascobolus (2), Neurospora (3), yeast (4), and Aspergillus (5). The high conversion end is usually the 5′ end (6), but can also be the 3′ end (7). Conversion frequency gradients are generally accepted to reflect a preferential initiation site for recombination that is located at the high conversion end of the gene (8, 9).In most organisms that undergo meiosis, only one of the four meiotic products is ordinarily recovered, so it is not possible to identify gene conversion on the basis of aberrant segregation ratios. A notable exception is the Arabidospsis quartet1 (qrt1) mutant that allows pollen tetrad analysis and has been used to demonstrate gene conversion events unambiguously by their classic 3:1 segregation (10) to estimate genome-wide conversion frequencies (11, 12) and to measure the tract lengths of such conversion events (12, 13). Usually, however, gene conversions have been identified by the flanking marker arrangement of intragenic recombinants (IGRs). This convention derives from the observation that, in yeast asci displaying gene conversion of a central marker flanked by two closely linked outside markers, the convertant spore could carry a parental or a recombinant arrangement of flanking markers with about equal frequency, on average (14, 15). Therefore, geneticists studying recombination in organisms where tetrad analysis is not possible have tended to refer to IGRs bearing a parental or noncrossover (NCO) arrangement of flanking markers as convertants, a convention that we also follow in this paper. In these cases, a convenient way of detecting conversion polarity is to compare the relative frequencies with which the two parentally marked IGRs are recovered from heteroallelic combinations (5, 16).In contrast to observations in fungi, it was noted repeatedly in maize recombination experiments that the vast majority of IGRs were crossovers (COs), i.e., associated with an exchange of flanking markers (17). Most of those studies dealt with polymorphic heterozygotes in which the recombining heteroalleles were derived from progenitor alleles that differed by single nucleotide and indel polymorphisms in as much as 1.6% of their sequences (18) and often included a transposon insertion allele. This experimental setup helped to place recombination junctions, but affected the outcome of the experiment. A different picture emerged from recombination studies with dimorphic heterozygotes at the bz locus, in which the recombining heteroalleles were derived from the same progenitor and differed only at the two sites between which recombination was being measured (19). In dimorphic heterozygotes, one CO class still predominated (that expected from the relative location of the mutations in the gene), but the NCO classes occurred in much higher numbers, so that the CO/NCO ratio was often less than 1. Similar results have been obtained at the r locus (20). Thus, the CO/NCO ratio in maize can be allele dependent. The variability in the CO/NCO ratio for different recombination hotspots observed in a highly polymorphic yeast hybrid (21) may reflect the extent of allelic polymorphisms among different loci.The sharp difference in the outcome of experiments involving polymorphic and dimorphic heterozygotes was explained in terms of the dual recombination pathway proposed by Allers and Lichten (22) and supported by other work (23–25), whereby repair of the initiating DSB produces COs via a double Holliday junction (DHJ) intermediate and NCOs via a synthesis-dependent strand annealing (SDSA) pathway. In this model, the decision to repair a DSB as a CO (via a DHJ) or a NCO (via SDSA) would happen at or soon after the initial step of strand invasion. The bz and r data suggest that that decision is affected by the extent of mismatches in the heteroduplex DNA formed by the invading strand. In the absence of extensive heterologies, mismatch repair proteins would not bind to the heteroduplex and the newly synthesized strand would be displaced readily leading to the recovery of NCO products.In the studies with bz dimorphic heterozygotes, the two NCO classes occurred in roughly similar numbers, i.e., the NCO class that carried the flanking markers of the proximal (5′) allele was recovered approximately as frequently as the NCO class that carried the flanking markers of the distal (3′) allele. Thus, there was no indication of preferential conversion of the proximal or distal allele (26, 27). However, the sample of mutant sites used in those studies did not include any at either end of the gene. Because most conversion gradients in yeast show strong 5′ or 3′ polarity, we could have missed a conversion gradient at bz that was steep at one or both ends but hardly detectable thereafter. To examine at greater depth the issue of conversion polarity within a higher plant gene, we now isolated a series of new bz mutations from the Bz-McC progenitor allele and extended our analysis to include sites at both the 5′ and 3′ ends of the bz gene. Mutations covering the length of the gene were tested in all possible pairwise combinations in two large diallels, with remarkably similar results in the two experiments. We find that bz mutants derived from the Bz-McC allele, which is flanked by single-copy DNA sequences on either side (28), show a U-shaped conversion gradient, with higher conversion frequencies at both the 5′ and 3′ ends than at the center. The implications of these findings are discussed with reference to recent data from genome-wide studies in several organisms.     

Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.