Liposomal FK506 is a new formulation of FK506 that increases FK506 levels in the liver and decreases them in the kidney in comparison to conventional IV formulation. In the present study, the efficacy of liposomal FK506 was evaluated in canine kidney and liver transplantation models. Liposomal FK506 increased the immunosuppressive efficacy of FK506 in the liver transplantation model, but decreased it in the kidney transplantation model. These results suggest that local immunosuppressive effects with increased intragraft FK506 level would play an important role in enhancing the immunosuppressive efficacy of liposomal FK506 in liver transplantation.

Animals↗

PubMedDate not supplied

High-yield affinity alkylation of the atrial natriuretic factor receptor binding site.

To facilitate characterization of the atrial natriuretic factor (ANF) receptor, we have developed an affinity labeling procedure, stepwise affinity labeling, which allows specific labeling of ANF binding sites in adrenal plasma membranes at high yields. An iodoacetyl (IAc-), bromoacetyl (BrAc-), or maleimidobenzoyl group was attached to the amino-terminal alpha-amino group of the ANF(4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, N4alpha-IAc-[125I]ANF(4-28), N4alpha-BrAc-[125I]ANF(4-28), and N4alpha-(maleimidobenzoyl)-[125I]ANF(4-28). Receptor labeling was carried out in a stepwise fashion as follows: (1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or N-ethylmaleimide to block sulfhydryl groups; (2) the affinity reagent was allowed to bind to the receptor at 0 degrees C for 1 h; and (3) the membranes were washed to remove unbound reagent and were incubated at room temperature to effect alkylation reaction. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography revealed specific labeling of a 130-kDa ANF receptor. On the basis of 125I-radioactivity incorporated, the labeling yields were estimated to be 70%, 52%, and 21% for the reactions with IAc-[125I]ANF(4-28), BrAc-[125I]ANF(4-28), and (maleimidobenzoyl)-[125I]ANF(4-28), respectively. The efficiency of receptor labeling by the stepwise procedure using IAc-[125I]ANF(4-28) was 27-fold greater than that obtained by photoaffinity labeling using N3Bz-[125I]ANF(4-28) and 63-fold greater than that by direct cross-linking using disuccinimidylsuberate and [125I]ANF(4-28) under comparable conditions. Digestion of the membrane protein labeled with IAc-[125I]ANF(4-28) by BrCN, endoproteinase Glu-C, and endoproteinase Lys-C gave single radiolabeled bands with apparent masses of 40, 18, and 29 kDa, respectively. Reversed-phase HPLC separation of the digests also gave single major peaks. The confinement of the affinity label to one major fragment in each digest suggests that the cross-linking occurred at a single or a limited number of sites. The stepwise affinity labeling with the high cross-linking yield and specificity may be useful for analyzing the ANF receptor binding site structure.

Adrenal Cortex↗

PubMedDate not supplied

Hippocampal damage in the human brain after cardiac arrest.

BACKGROUND AND PURPOSE: Very few reports are available on changes in the human hippocampus after cardiac arrest. The objective of this study was to investigate if specific hippocampal volume losses can be demonstrated in the human brain following reperfusion after cardiac arrest. METHODS: We assessed the volumes of the hippocampal formation (HF) and temporal lobe excluding HF (TL) as the contrast using magnetic resonance (MR)-imaging-based volumetry in 11 vegetative patients after cardiac arrest and in 22 healthy controls of similar age, sex and body size distribution. The measured volumes were normalized for differences in the head size among subjects by dividing by the total intracranial volume (TICV). The MR images of the 11 patients were obtained between days 8 and 21 after cardiac arrest. RESULTS: The observed volumes of HFs and TLs of both patient and control groups were as follows: right HF volume (HFV): 2.67 +/- 0.19 (mean +/- SD, cm(3)) in patients versus 3.89 +/- 0.44 in controls; left HFV: 2.72 +/- 0.17 versus 3.74 +/- 0.35; right TL volume (TLV): 73.37 +/- 6.54 versus 80.08 +/- 7.62, and left TLV: 72. 45 +/- 6.77 versus 78.59 +/- 6.68. The normalized indices (HFV/TICV and TLV/TICV) were as follows: right HF: 0.0021 +/- 0.0002 (mean +/- SD) in patients versus 0.0031 +/- 0.0001 in controls, p < 0.0001, left HF: 0.0022 +/- 0.0002 versus 0.0030 +/- 0.0001, p < 0.0001, right TL: 0.058 +/- 0.002 versus 0.064 +/- 0.004, p = 0.0007, and left TL: 0.058 +/- 0.002 versus 0.062 +/- 0.004, p = 0.0014. The HFV-TLV ratios (HFV/TICV divided by TLV/TICV) of both groups were: right HFV-TLV ratio: 0.037 +/- 0.004 in patients versus 0.049 +/- 0. 004 in controls, p < 0.0001, left HFV-TLV ratio: 0.038 +/- 0.004 versus 0.048 +/- 0.004, p < 0.0001. CONCLUSIONS: The patient group had HFs that were 26.8-30.6% smaller than those of the control group, but in the patient group, the TLs slightly decreased in size by only 7.8-8.2% of the volume of those in the control group within 21 days after cardiac arrest. The volume reductions in the bilateral HFs of patients after cardiac arrest were significantly larger than those in the bilateral TLs. We speculate that this specific rapid hippocampal shrinkage reflects its greater vulnerability to global brain ischemia.

Aged↗

PubMedDate not supplied

Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells.

Caspase-3 (CPP32/Yama/apopain), one of the interleukin 1 -converting enzyme (ICE)-like proteases (caspases), is anticipated to mediate apoptotic cell death. We observed the expression of caspase-3 in various cancer cell lines and lack of normal expression of mRNA and protein in MCF-7, human breast carcinoma cell line. Sequence analysis of cDNA showed 125 nucleotides deletion in spite of no gross gene alteration of caspase-3 in MCF-7. The possible cause is altered splicing of the fragment followed by frame shift at translation level. MCF-7 cells are widely used in the research of apoptosis because of the high sensitivity to tumor necrosis factor induced cell death. However, our results suggest the existence of other apoptotic pathways independent on caspase-3 at least in MCF-7 cells.

Adenocarcinoma↗

PubMedDate not supplied

Increased expression levels of cyclin-dependent kinase inhibitor p27 correlate with good responses to platinum-based chemotherapy in non-small cell lung cancer.

In order to determine whether expression of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer (NSCLC) correlates with chemotherapeutic response, resected tumors from 22 patients with recurrent lung cancer who had undergone complete resection and received chemotherapy after the initial tumor recurrence were subjected to p27 immunostaining. Histological examination of the resected tumors revealed 14 adenocarcinomas, 7 squamous cell carcinomas and one adenosquamous cell carcinoma. Fifty percent or less and over 50% of the cells in the resected tumors of 11 patients each (groups 1 and 2, respectively) were p27-immunopositive. All but one patient received platinum-based chemotherapy after recurrence. Only one in group 1 achieved a partial response (PR) in chemotherapy whereas 2 and 4 in group 2 achieved complete and PRs, respectively. The chemotherapy response rate of group 2 (54%) was significantly higher than that of group 1 (9%, p=0.022). The times to recurrence after tumor resection of the 2 groups did not differ significantly (log-rank p=0.23, Wilcoxon p=0. 32), but survival after chemotherapy of group 2 was significantly better than that of group 1 (log-rank p=0.045, Wilcoxon p=0.028). It is suggested that high p27 expression levels in tumors may predict the good responses to platinum-based chemotherapy for NSCLC.

Antineoplastic Combined Chemotherapy Protocols↗

PubMedDate not supplied

Increased expression levels of p53 correlate with good response to cisplatin-based chemotherapy in non-small cell lung cancer.

In order to determine whether expression of the tumor suppressor gene p53 in non-small cell lung cancer (NSCLC) correlates with chemotherapeutic response, resected tumors from 18 patients with recurrent lung cancer who had undergone complete resection and received chemotherapy after the initial tumor recurrence were subjected to p53 immunostaining. Histological examination of the resected tumors revealed 11 adenocarcinomas, 6 squamous cell carcinomas and one adenosquamous cell carcinoma. Group 1 was </=50% (n=9) and group 2 >50% (n=9) p53-immunopositive. All patients received cisplatin-based chemotherapy after recurrence. No patient in group 1 achieved response to chemotherapy whereas 2 and 3 in group 2 achieved complete and partial responses, respectively. The chemotherapy response rate of group 2 (56%) was significantly higher than that of group 1 (0%, p=0.009). The times to reoccurrence after tumor resection of group 2 was significantly better than that of group 1 (log-rank p=0.019, Wilcoxon p=0.042), and survival after chemotherapy of group 2 was also significantly better than that of group 1 (log-rank p=0.023, Wilcoxon p=0.034). It is suggested that high p53 expression levels in tumors correlate with both good response to cisplatin-based chemotherapy and good survival of patients with advanced NSCLC.

Aged↗

PubMedDate not supplied

Upregulated expression of angiogenesis genes and down regulation of cell cycle genes in human colorectal cancer tissue determined by cDNA macroarray.

The differential expression of hundreds of tightly, transcriptionally controlled genes in isolated human colorectal cancer and respective normal mucosa from two patients was analyzed by the cDNA macroarray technique. mRNA prepared from the colorectal cancer tumors was compared with 588 genes spotted onto the filter. Case A showed down-regulation of the expression of cell-cycle-related genes including cyclins, cyclin-dependent kinase (CDK) 2, and CDK-activating kinase, as compared with normal mucosa from the same patient. The tumors showed up-regulation of expression of angiogenesis-related genes such as type II cytoskeletal 8 keratin, metalloproteinase subtypes, VEGF, and bFGF, to over 5-fold the levels in normal mucosa. Thus, colorectal carcinoma tissues are characterized by the upregulation of molecules related with angiogenesis. These results suggest that angiogenesis-related molecules are suitable candidates for target-based therapies for colorectal cancer patients. In case B, the largest difference in expression between the tumor and mucosal tissues was observed in the MMP-1 gene. In contrast to the first case, there was no increase in expression of angiogenesis-related molecules or decrease in expression of cell-cycle-regulatory molecules. The expression profile was quite different between these two patients. This approach may eventually provide a mean of selecting target-based drugs in individual colon cancer patients.

Adult↗

PubMedDate not supplied

Effect of taxol on vinblastine sulfate-induced crystallization of tubulin.

In human lung adenocarcinoma cells exposed to vinblastine (VLB) and stained with fast green, large tubulin crystals were seen in the cytoplasm. In contrast cells exposed to taxol had prominent bundles of stabilized microtubules. Pre- or co-incubation of cells with taxol before exposure to VLB prevented formation of the crystals. However, VLB did not prevent formation of stabilized microtubules by taxol if added at the same time. Under a fluorescent microscope, VLB induced crystals stained with secondary antibodies against anti-alpha- and anti-beta- tubulin antibodies. Neither of the drugs altered binding of the antibodies to these crystals.

Adenocarcinoma↗