Efficient, Repeated Adenovirus-Mediated Gene Transfer in Mice Lacking both Tumor Necrosis Factor Alpha and Lymphotoxin α

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα^−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα^−/− and TNF-α/LTα^+/− mice versus TNF-α/LTα^+/+ mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111.

An extracellular alkaline lipase of alkalophilic Pseudomonas pseudoalcaligenes F-111 was purified to homogeneity. The apparent molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000, and the isoelectric point was 7.3. With p-nitrophenyl esters as its substrates, the enzyme shows preference for C12 acyl and C14 acyl groups. It was stable in the pH range of 6 to 10, which coincides with the optimum pH range.     

Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly.

Hepatitis delta virus (HDV) contains two virus-specific delta antigens (HDAgs), large and small forms, which are identical in sequence except that the large one contains 19 extra amino acids at the C terminus. HDAgs are nuclear phosphoproteins with distinct biological functions; the small form activates HDV RNA replication, whereas the large form suppresses this process but is required for viral particle assembly. In this study, we have characterized the phosphorylative property of HDAg in a human hepatoma cell line (HuH-7) and examined the role of phosphorylation in HDAg function. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation levels of both HDAgs were diminished by the inhibitor of casein kinase II (CKII). Nevertheless, phosphorylation of only the small form could be markedly reduced by the protein kinase C (PKC) inhibitor, suggesting different phosphorylation properties between the two HDAgs. When these two kinase inhibitors were added separately to the transient-expression system, HDV RNA replication was profoundly suppressed. In contrast, the inhibitors did not affect the assembly of empty HDAg particle from HDAgs and hepatitis B virus surface antigen. To further examine the role of phosphorylation in HDAg function, two conservative CKII recognition sites at Ser-2 and Ser-123 of both HDAgs and one potential PKC recognition site at Ser-210 of the large HDAg were altered to alanine by site-directed mutagenesis. Transfection experiments indicated that mutation at Ser-2, but not Ser-123, significantly impaired the activity of the small HDAg in assisting HDV RNA replication. This property is in accordance with our observation that Ser-2, not Ser-123, was the predominant CKII phosphorylation site in the small HDAg. Our studies also excluded the possibility that the phosphorylation of Ser-2, Ser-123, or Ser-210, had roles in the trans-suppression activity of the large HDAg, in the assembly of empty virus-like HDAg particle, and in the nuclear transport of HDAgs. In conclusion, our results indicate that both CKII and PKC positively modulate HDV RNA replication but not the assembly of empty HDAg particle. The role of CKII in HDV replication may at least in part be accounted for by the phosphorylation of Ser-2 in the small HDAg. The effect of PKC on HDV RNA replication is, however, not to mediate the phosphorylation of the conservative Ser-210 in the large HDAg but rather to act on as-yet-unidentified Ser or Thr residues in the small HDAg or cellular factors. These findings provide the first insight into the roles of phosphorylation of the two HDAgs in the HDV replication cycle.     

Competing risk analysis for life table data with known observation times

"Several models have been proposed for the analysis of cohort mortality in the presence of competing risks.... This paper describes a maximum likelihood approach to the analysis of follow up data in life table format for the case of two competing risks--a specific cause and its competing complement. The model developed uses a robust survivorship assumption--the piecewise exponential--and takes into account information on time to death and time to withdrawal." (summary in GER)     

Changes in Lipogenic Capacity and Activities of Ketolytic and Lipogenic Enzymes in Brain Regions of Developing Rats

Oxidation of ketone bodies (KBs) generates acetyl coenzyme A (AcCoA), which can be further incorporated into fatty acid. We have determined the rates of lipogenesis from ketone bodies in developing rats and their relation to the activities of enzymes involved in the production of cytoplasmic AcCoA via different pathways in brain regions. In the cerebrum (Cbr), rates of fatty acid synthesis from [3-14C]acetoacetate ([3-14C]AcAc) were high during the early postnatal period but decreased rapidly thereafter until weaning. Although similar developmental patterns of synthesis characterized the cerebellum (Cbl), midbrain (Mb), brain stem (Bs), and thalamus (Th), maximal rates were highest in the Cbr and lowest in the Th. In all regions, synthetic rates were higher throughout the entire suckling period than in adulthood. There were not appreciable differences in synthetic rates among brain regions of adult rats. The developmental changes in rates AcAc incorporation into fatty acids were closely related to AcAcCoA synthetase activity, but not to activities of ATP-citrate lyase or AcCoA synthetase. During the early postnatal stage enhanced rates of lipogenesis were accompanied by increased activities of AcAcCoA synthetase in all regions, with the highest activity occurring in the Cbr. The sequence of reactions coupling AcAcCoA synthetase and AcAcCoA thiolase in cytoplasm may be an important pathway for generation of AcCoA from KBs for fatty acid synthesis in all regions of the developing brain. This interpretation is strengthened by evidence of concomitant increases in the activities of fatty acid synthetase and AcCoA carboxylase.     

Current-dependent block of endplate channels by guanidine derivatives

Methyl- and ethylguanidine block the endplate current in frog muscle. Both derivatives blocked inward-going endplate currents without affecting outward endplate currents. Repetitive stimulation that evoked several inward endplate currents enhanced the block, which suggests that these agents interact with open endplate channels. The relative conductance vs. potential curve exhibited a transition from a low to a high value near the reversal potential for the endplate current, both in normal and in 50% Na solution. In the latter solution, the reversal potential for endplate current was shifted by a mean value of 16 mV in the direction of hyperpolarization. The results suggest that methyl- and ethylguanidine block open endplate channels in a manner dependent on the direction of current flow rather than on the membrane potential.     

人淋巴细胞的体外抗体诱发及其在杂交瘤中的应用

本文应用手术切除的人扁桃体或外伤脾为材料,在体外诱发了针对不同类型抗原的抗体应答,其中包括羊红细胞、卵白蛋白、破伤风类毒素和乙肝病毒表面抗原的抗体应答,并应用破伤风类毒素体外激活的人B淋巴细胞制作杂交瘤。根据多次融合的结果表明其融合率平均为52.1%,抗体阳性率为15.6%,分泌IgM和IgG抗体的阳性集落孔的比例接近1∶1。我们还研究了体外诱发人淋巴细胞产生抗体的条件,发现细胞类群(在本研究中是采用不同的细胞分离方法)是主要的影响因素,值得进一步研究。目前所建立的体外免疫实验系统,主要是应用尼龙毛柱法富集B细胞,在96孔培养板上加上一定剂量范围的抗原刺激。为了协助淋巴细胞反应,可再加10%的T细胞条件培液或少量丝裂原混合物(LPS0.1μg/ml+PWM1μg/ml)。     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

Joint estimation of immigration and mating system parameters in gymnosperms using the EM algorithm

Summary An EM algorithm procedure is presented for the maximum-likelihood joint estimation of immigration and mating system parameters of mixed-mating system models for gymnosperms. In addition to accommodating multiallelic and multilocus data in mature populations and pollen pools, the EM estimates are insensitive to allelic frequency changes in foreign population and may approach closer to global maximum-likelihood estimates with each iteration, regardless of initial starting values. Estimates of rates of selfing (), outcrossing (Ô), and immigration (Î) derived from the model are bounded strictly within the natural biological range (i.e., 0 Ô + Î 1; + Ô + Î = 1).     

The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase

We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli. The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map. We cloned the gene in a high copy number vector and purified the enzyme. It is a monomer, with an apparent molecular weight of 65,000. The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose. We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates. The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin. In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose. To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation. More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose. Furthermore, [14C]maltotriose, specifically labeled at the reducing end, was hydrolyzed to [14C]glucose and unlabeled maltose. These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides. The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960. Homology to glucosidases, alpha-amylases, and pullulanases were observed. Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.