组织型纤溶酶原激活剂（t—PA）工程细胞系遗传特性及成瘤性分析

组织型纤溶酶原激活剂(t-PA)因其在防止血栓形成中起重要作用而受到人们的重视。但由于t-PA在血液中半衰期很短,作为溶栓药,一时难于推广。为了延长半衰期、增强其特异活性,本组构建了t-PA突变体并在CHO-dhfr~-细胞中获得了高效表达。我们在细胞培养基中加入秋水仙素,通过低张、固定、染色,进行染色体分析,结果表明,t-PA工程细胞系染色体条数为20条,畸变类型有异着丝粒。四倍体、裂隙、断片,畸变率为15％,属于正常范围。同时我们对该细胞系进行成瘤性试验,选用4周龄裸鼠作为试验鼠,以Hela细胞为阳性对照,CHO-dhfr~-细胞为阴性对照,试验表明:t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

Flooding-induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves

Flooding effects on membrane permeability, lipid peroxidation and activated oxygen metabolism in corn (Zea mays L.) leaves were investigated to determine if activated oxygens are involved in corn flooding-injury. Potted corn plants were flooded at the 4-leaf stage in a controlled environment. A 7-day flooding treatment resulted in a significant increase in chlorophyll breakdown, lipid peroxidation (malondialdehye content), membrane permeability, and the production of superoxide (O ₂ ^- ) and hydrogen peroxide (H₂O₂) in corn leaves. The effects were much greater in older leaves than in younger ones. Spraying leaves with 8-hydroxyquinoline (an O ₂ ^- scavenger) and sodium benzoate (an .OH scavenger) reduced the oxidative damage and enhanced superoxide dismutase (SOD) activity. A short duration flooding treatment elevated the activities of SOD, catalase, ascorbate peroxidase (AP), and glutathione reductase (GR), while further flooding significantly reduced the enzyme activities but enhanced the concentrations of ascorbic acid and reduced form glutathione (GSH). It was noted that the decline in SOD activity was greater than that in H₂O₂ scavengers (AP and GR). The results suggested that O ₂ ^- induced lipid peroxidation and membrane damage, and that excessive accumulation of O ₂ ^- is due to the reduced activity of SOD under flooding stress.     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Repression by a differentiation-specific factor of the human cytomegalovirus enhancer.

We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.     

On the existence of stable pairing distributions

We formulate and analyze pair-formation models for multiple groups with general pairing rates and arbitrary mixing probabilities. Under the assumption of constant recruitment rates and equal average duration of all types of partnerships, we have shown that the dynamics are relatively simple because of the monotonicity properties of the dynamical system associated with the pairing/mixing of heterogeneous populations of male and female individuals. In fact, we have shown that the corresponding asymptotic stable paired distribution is given precisely by the asymptotic values of the matrices that prescribe the mixing/contact structure. In other words, if the sizes of the mixing subpopulations of males and females are asymptotically constant and if the average durations of partnerships are about the same regardless of type, then the matrices that describe the mixing between subpopulations also characterize the distribution of paired types. Alternatively, if the distribution of the average duration of relationships between individuals has a large variance then it may be impossible to detect any relationship between the mixing/contact structure and the observed distribution of paired types. The study of models with constant per-capita recruitment rates give rise to homogeneous systems of degree one. The analysis of the dynamics of pairs for models with exponentially growing populations of singles is complicated. So far, we are only able to classify the stability of all non-strictly positive boundary exponential solutions. From our incomplete analysis, it is not possible to detect necessary and sufficient conditions for the existence and stability of strictly interior exponential solutions. We cannot rule out the possibility of oscillations. The mathematical problems associated with the stability of exponential solutions of dynamical systems of degree one are of relevance in demography, epidemiology, and population dynamics.On leave from University of Alabama in Huntsville     

Molecular definition of red cell Rh haplotypes by tightly linked SphI RFLPs.

The Rh blood group system of human red cells contains five major antigens D, C/c, and E/e (the latter four designated "non-D") that are specified by eight gene complexes known as Rh haplotypes. In this paper, we report on the mapping of RH locus and identification of a set of SphI RFLPs that are tightly linked with the Rh structural genes. Using exon-specific probes, we have localized the SphI cleavage sites resulting in these DNA markers and derived a comprehensive map for the RH locus. It was found that the SphI fragments encompassing exons 4-7 of the Rh genes occur in four banding patterns or frameworks that correspond to the distribution and segregation of the common Rh haplotypes. This linkage disequilibrium allowed a genotype-phenotype correlation and direct determination of Rh zygosity related to the Rh-positive or Rh-negative status (D/D, D/d, and d/d). Studies on the occurrence of SphI RFLPs in a number of rare Rh variants indicated that Rh phenotypic diversity has taken place on different haplotype backgrounds and has arisen by diverse genetic mechanisms. The molecular definition of Rh haplotypes by SphI RFLP frameworks should provide a useful procedure for genetic counseling and prenatal assessment of Rh alloimmunization.     

Simultaneous Determination of Dermatan Sulfate and Oversulfated Dermatan Sulfate in Plasma by High-Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization

A chemical method for the determination of dermatan sulfate (DS) and oversulfated dermatan sulfate has been developed and applied to the pharmacokinetic study of these polysaccharides in experimental animals. The analytical procedure includes a simple preparation step of administered DS and oversulfated DS from blood plasma, HPLC for the separation and detection of DS and oversulfated DS using an Asahipak NH₂P-50 column, fluorometric reaction of the polysaccharides with guanidine in a strong alkaline medium. DS and oversulfated DS were extracted from plasma by treating it with proteinase to remove plasma proteins and recovered with endogenous plasma glycosaminoglycans by ethanol precipitation. Finally, DS and oversulfated DS were analyzed by fluorometric HPLC. The detection limits of DS and oversulfated DS were 10 and 20 ng, respectively. Furthermore, we demonstrated that artificial oversulfation of DS increased its biological half-life after intravenous administration to rats.     

Heterotropic effects of chloride on the ligation microstates of hemoglobin at constant water activity.

Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.