In vivo delivery of interferon-α gene enhances tumor immunity and suppresses immunotolerance in reconstituted lymphopenic hosts.

T cells recognize tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP); however, HP-driven antitumor responses gradually decay in association with tumor growth. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study we examined whether a tumor-specific immune response induced by IFN-α could enhance and sustain HP-induced antitumor immunity. An intratumoral IFN-α gene transfer resulted in marked tumor suppression when administered in the early period of syngeneic hematopoietic stem cell transplantation (synHSCT), and was evident even in distant tumors that were not transduced with the IFN-α vector. The intratumoral delivery of the IFN-α gene promoted the maturation of CD11c(+) cells in the tumors and effectively augmented the antigen-presentation capacity of the cells. An analysis of the cytokine profile showed that the CD11c(+) cells in the treated tumors secreted a large amount of immune-stimulatory cytokines including interleukin (IL)-6. The CD11c(+) cells rescued effector T-cell proliferation from regulatory T-cell-mediated suppression, and IL-6 may have a dominant role in this phenomenon. The intratumoral IFN-α gene transfer creates an environment strongly supporting the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the induction of tumor-specific immunity and suppression of immunotolerance.

Comprehensive survey of proteins targeted by chloroplast thioredoxin.

Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH(2)-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.

Preoperative staging of rectal cancer using a 7.5 MHz front-loading US probe.

BACKGROUND: Conventional echoendoscopes have disadvantages when used for staging colorectal cancer including the inability to pass the instrument through tight stenosis and limited maneuverability. This study evaluated the preoperative use of a newly developed 7.5 MHz front-loading ultrasound probe (FLUP) for local staging of rectal cancer. METHODS: A 7.5 MHz FLUP, diameter 7.3 mm, was used in this study. The mechanical shaft portion of the probe can be passed in retrograde fashion through the accessory channel of a standard colonoscope. Thirty-nine patients with rectal cancer underwent ultrasonography with this probe. The tumors were staged using the TNM system, and the results were compared with the histologic findings of the resected specimens. RESULTS: The FLUP proved to be satisfactory, with respect to maneuverability, for traversing stenosis and accurate recognition of small tumors under direct endoscopic control. The accuracy of the FLUP for T staging was 82% (32 of 39) for all tumors, 90% in pT1, and 79% in pT2 to pT4 tumors. The accuracy of the FLUP for N staging was 72% (23 of 32) overall. The sensitivity was 83%, the specificity was 65%, the positive predictive value was 59%, and the negative predictive value was 87%. CONCLUSIONS: The 7.5 MHz FLUP appears to be useful for preoperative local staging of rectal cancer. This system makes it technically easier to image small cancers as well as advanced rectal cancers.

The gamma subunit in chloroplast F(1)-ATPase can rotate in a unidirectional and counter-clockwise manner.

Rotation of the gamma subunit in chloroplast F(1)-ATPase (CF(1)) was investigated by using a single molecule observation technique, which is developed by Noji et al. to observe the rotation of a central gamma subunit portion in the alpha(3)beta(3)gamma sub-complex of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) during ATP hydrolysis [Noji, H. et al. (1997) Nature 386, 299-302]. We used two cysteines of the gamma subunit (Cys-199 and Cys-205) of CF(1)-ATPase, which are involved in the regulation of this enzyme, to fix the fluorochrome-labeled actin filament. Then we successfully observed a unidirectional, counter-clockwise rotation of the actin filament with the fluorescent microscope indicating the rotation of the gamma subunit in CF(1)-ATPase. We conclude that the rotation of the gamma subunit in the F(1)-motor is a ubiquitous phenomenon in all F(1)-ATPases in prokaryotes as well as in eukaryotes.

The S29 ribosomal protein increases tumor suppressor activity of K rev-1 gene on v-K ras-transformed NIH3T3 cells.

The human S29 ribosomal protein (S29 rp) cDNA has been isolated from differential hybridization screening of a colon carcinoma cDNA library. Northern blot analysis showed that the level of S29 rp mRNA was higher in undifferentiated HT29 human colon carcinoma cells than in a morphologically differentiated subclone under the same growth condition. Furthermore, the level of S29 rp mRNA was downregulated in rapidly proliferating HT29 cells, as compared to the contact inhibited cells. Interestingly, the amount of Krev-1 mRNA was inversely correlated with respect to the amount of S29 rp mRNA in these cells. To examine a functional link between S29 rp and Krev-1 protein, we co-transfected the expression vectors containing wild-type or mutant S29 rp and mutationally activated Krev-1(63E) cDNAs into the v-Ki-ras-transformed NIH3T3 (DT) cells, and observed the induction of flat revertants. Krev-1(63E) induced a certain amount of flat colonies, while S29 rp alone also induced flat colonies at low frequencies. Interestingly, revertant-inducing activity of Krev-1(63E) was significantly enhanced by S29 rp. We have also demonstrated that a zinc finger-like domain of S29 rp indeed has a zinc binding activity and a derivative, S29 rp(ms), which was unable to bind zinc ion but still retained revertant inducing activity by itself, could not functionally interact with Krev-1(63E) protein.

[Primary intramedullary melanocytoma of medulla oblongata: a case report].

A case of intramedullary melanocytoma of the medulla oblongata; which has not been hitherto reported, is presented. The tumor ran on a slow progressive course. The patient died of pneumonia seven months after the operation. MRI findings did not specify this tumor as they were not distinct from those of malignant melanoma. The tumor was totally black and was not attached to the dura. Microscopically, the neoplastic cells were uniform in size and possessed abundant melanin granules in their cytoplasm. Mitosis was very rare. The ultrastructure of the tumor cells showed many melanosomes at varying stages of maturation. The melanocytoma in the eloquent region has an unfavorable outcome.

Refolding proteins by gel filtration chromatography.

We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonuclease A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of milligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.

Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

c-Ets-1 protein is hyperphosphorylated during mitosis.

The ets-1 and ets-2 proto-oncogene products can serve as transcription factors and become phosphorylated in response to Ca(2+)-mediated signals. We have examined expression of Ets proteins during the cell cycle in cells synchronized by centrifugal elutriation or nocodazole-induced mitotic block. Both methods revealed the presence of a hyperphosphorylated isoform of Ets-1 during the mitotic phase. This isoform showed a characteristic mobility shift and was observed during mitosis in each of four cell lines (three human T-cell lines and a human astrocytoma) that express ets-1. In elutriated cells, only a small portion of the Ets-1 in cells from the G2/M fractions was hyperphosphorylated, while in nocodazole-arrested cells more of the Ets-1 was shifted. When cells were released from nocodazole arrest, this isoform disappeared within 1-2 h as cells completed mitosis and entered G1. This suggests that hyperphosphorylated Ets-1 is present transiently during early mitosis, before or around the time of the metaphase-anaphase transition. Exposure of unsynchronized cells to okadaic acid resulted in a dramatic hyperphosphorylation of virtually all Ets-1, suggesting that changes in cellular phosphatase activity are important for cell cycle regulation of Ets-1. Hyperphosphorylated Ets-1 appears to arise from multiple phosphorylations on serine in the exon 7-encoded domain of the protein and did not appear to alter sequence-specific DNA-binding activity. Although enhanced phosphorylation of Ets-2 was detected in nocodazole-arrested cells, no Ets-2 hyperphosphorylation was seen.

[Long-term follow up of trigeminal neuralgic patients treated by retrogasserian rhizotomy or by microvascular decompression].

The authors studied the long-term follow up of trigeminal neuralgic patients treated by retrogasserian rhizotomy with anhydrous glycerol injection (GI) or by microvascular decompression (MVD) and compared the satisfaction rates between them. Since 1983, 67 patients had been treated either by GI or MVD. The authors analyzed the results of the treatment by the method of questionnaire. Fifty-two patients of these 67 answered the questions, on which the present analysis was based. GI group consisted of 27 patients and MVD group 29 patients. The average follow-up period was 30.9 months in GI group (maximum follow-up: 84 months) and 50.9 months in MVD group (maximum follow-up: 95 months), respectively. The technique of GI used was Härtel's method with cisternography of Meckel's cave. The operations of MVD were performed by J.H. and A.K.. One patient out of 29 failed to be treated by MVD and two recurred within one year. On the other hand, 2 patients out of 27 failed to be treated by GI and 11 recurred. The recurrence was seen earlier in patients who had undergone GI, while one patient recurred the neuralgia at 76 months later. The recurrence rate in patients treated by MVD is 7.0% at 95 months, while that by GI is 49.0% at 84 months based on Kaplan-Meier survival analysis. The pathogenesis of trigeminal neuralgia is speculated to be an emphatic conduction caused by segmental demyelination and artificial synapse formation at the junction of central and peripheral myelin. The factors of this demyelination may be multiple sclerosis, basilar impression, aneurysm, arteriovenous malformation, atheroscleroses and natural aging.(ABSTRACT TRUNCATED AT 250 WORDS)

[Preliminary clinical trial of intrathecal rt-PA (TD-2061) for the prevention of cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage].

The results of preliminary clinical trial (a multicenter, open-label, dose escalation study) of intrathecal recombinant tissue plasminogen activator (rt-PA) for the prevention of cerebral vasospasm were reported. Seventeen patients admitted within 48 hours of subarachnoid hemorrhage (SAH) were enrolled in this study. Patients ranged from 42 to 69 years of age. All cases enrolled were classified in clinical grade II, III or IV according to the classification of Hunt and Kosnik and in group 3 or 4 according to Fisher's CT grading scale. Surgery for clipping the aneurysms were performed and a small silicone catheter was left in the subarachnoid space. Twenty four hours after the surgery intrathecal bolus infusion of rt-PA was started through the silicone catheter at 6-hour intervals for 3 days. Patients were divided into 4 groups based on the dosage of rt-PA for each infusion. The dosage of rt-PA for each infusion and a number of cases in each group was as follows; 25 KIU in 4 cases, 75 KIU in 6 cases, 200 KIU in 4 cases and 600 KIU in 3 cases. There was no significant difference in the clearance of subarachnoid clots between four groups. However, the occurrence of both symptomatic and angiographic vasospasm was less in the 75 KIU group than in other three groups. Intracranial bleeding complications were noted in 4 patients (1/6 in the 75 KIU group, 2/4 in the 200 KIU group and 1/3 in the 600 KIU group). Serial coagulation studies demonstrated no evidence of systemic fibrinolysis. Disorientation was noted in 2 out of 3 patients of the 600 KIU group.(ABSTRACT TRUNCATED AT 250 WORDS)

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif.

ETS1 protein purified from CEM cells was used to select its optimum DNA-binding sequence (pu) G/CCaGGA-AGTc (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility-shift assays (EMSA) indicated that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity (Ka = 0.5-4.0 x 10(10) M-1) and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more ETS1-3 than PEA3. Competitive binding experiments showed that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, changing the core DNA-binding sequence from GGAA to AGAA eliminates competition. Since the human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides as did the Drosophila E74A protein (one of the most divergent members of the ETS family), this strongly suggests that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.