TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic ß Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet ß cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic ß cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-ß1, TNFα, and IL-1ß. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

Clinical effectiveness of 3 days preoperative treatment with recombinant human erythropoietin in total knee arthroplasty surgery: a clinical trial.

AIMS: The purpose of study is to evaluate the effect and complication of preoperative short-term daily recombinant human erythropoietin (rhEPO) treatment for blood-saving in patients undergoing unilateral primary total knee arthroplasty (TKA). METHODS: This three-arm randomized clinical trial compared three different rhEPO-based treatment protocols for unilateral primary TKA. Group A: application of daily doses of rhEPO combined with iron supplement starting 3 days before surgery; Group B: application of daily doses of rhEPO combined with iron supplement starting the day of surgery; Group C: iron supplement alone. Perioperative hemoglobin (Hb) level gaps, total perioperative blood loss, reticulocyte levels and treatment-related complications were studied. RESULTS: A total of 102 patients were included (35, 35 and 32 patients in Groups A, B and C, respectively). Total blood loss (TBL) in Groups A, B and C was 490.84, 806.76 and 924.21 ml, respectively. Patients in Group A had a significant lower TBL than Groups B and C (A vs. B: P = 0.010; A vs. C: P < 0.001). There was no difference as for TBL between Groups B and C (P = 0.377). Group A patients had significant smaller Hb decline than Group C on the third and fifth postoperative day (P = 0.049, P = 0.037), as well as than Group B on the fifth postoperative day (P = 0.048). There was no difference as for Hb decline between Groups B and C. No difference was shown in levels of inflammatory biomarkers or blood-saving protocol-related complications among three groups. CONCLUSIONS: Daily dose of rhEPO combined with iron supplement administered 3 days before TKA procedures could significantly decrease perioperative blood loss and improve postoperative Hb levels, without significantly elevating risks of complication, when compared with admission of rhEPO on the day of surgery and iron supplement alone. Preoperative daily rhEPO treatment could be a more effective blood-saving protocol in TKA procedures.

Degradation and dissolution of hemicelluloses during bamboo hydrothermal pretreatment.

To elucidate the hemicelluloses degradation and dissolution during hydrothermal pretreatment, hemicelluloses separated from both hydrolysate and pretreated substrate were investigated. Along with the pretreatment proceeding, some hemicelluloses fractions dissolved and diffused into the bulk liquor; MW (molecular weight) of these hemicelluloses fractions increased first and then decreased as well as amount of the fractions. Based on the definition of MW of the soluble hemicellulose, it has been concluded that some insoluble hemicellulose fractions appeared in the hydrolysate. In contrast, the hemicellulose degradation occurred continually and had been observed by the gradual decrease of MW of the hemicellulose isolated from pretreated substrate. Lingering dissolution at the later stage might attribute to the facts that some soluble fractions were still entrapped in the substrate. 5-15% hemicellulose fractions remained in the pretreated substrate at the later stage were composed of soluble species.

Lignin removal and benzene-alcohol extraction effects on lignin measurements of the hydrothermal pretreated bamboo substrate.

Lignin content of hydrothermal pretreated bamboo chips was determined by the two methods: TAPPI standard method (222om-06) and TAPPI standard method without benzene-alcohol extraction (BAE). The results showed that including BAE resulted in lower Klason lignin (KL) and acid soluble lignin (ASL) measurements in the prehydrolyzed substrate, that is to say, BAE removed parts of KL and ASL. Therefore, the TAPPI standard method should be modified by omitting the BAE for lignin measurements of pretreated substrate. The following lignin removal analysis suggested that lignin was removed from the bamboo substrate during pretreatment by a combination of degradation reaction and deconstruction; thereafter the pseudo lignin generated in the hydrothermal pretreatment and condensation reaction between the lignin fragments accounted for the later KL increase.

Hydrothermal pretreatment of bamboo and cellulose degradation.

A systematic hydrothermal pretreatment of bamboo chips had been conducted with an aim to trace the cellulose degradation. The results showed that cellulose chain cleavage basically occurred when the temperature exceeded 150°C. A slightly higher DP (degree of polymerization) than starting material had been observed at low temperature pretreatment. Treatment at higher temperature (≥ 170°C) caused severe cleavage of cellulose and therefore gave rise to low DP with more soluble species. DP of cellulose declined drastically without additional hemicelluloses dissolution when hemicelluloses removal reached to the limit level. Cellulose degradation under hydrothermal pretreatment generally followed the zero reaction kinetics with the activity energy of 121.0 kJ/mol. Besides, the increase of cellulose crystalline index and the conversion of Iα-Iß had also observed at the hydrothermal pretreatment.

Surface characterizations of bamboo substrates treated by hot water extraction.

Environment Scanning Electron Microscopy (ESEM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical changes on both the interior and exterior surface of bamboo (Dendrocalamopsis oldhami) substrates treated by hot water extraction. ESEM results showed the visible changes between exterior and interior surface of the treated substrates, in where spherical droplets did not extensively appear on both the surfaces at start of the pretreatment; nevertheless the droplets formation on the exterior surface occurred more rapidly than that of the interior surface. Results from XPS examination that the increase of C1 (C-C, C-H) concentration and decrease of O/C ratio and O1 (C=O) concentration of the samples on the both surfaces further demonstrated that both surfaces consisted of increasing amount of lignin as the extraction continued, especially for exterior surface. The O/C ratios finally reached to a level-off value with exterior surface 0.34 and interior surface 0.37.

The exogenous wild-type p14ARF gene induces growth arrest and promotes radiosensitivity in human lung cancer cell lines.

The cyclin-dependent kinase inhibitor p16INK4a encoded by the INK4A/CDKN2A/MTS1 gene is a frequent target of 9p21 inactivation in human lung cancers. The p14ARF transcript, which is an alternative spliced form of this locus, is also altered or deleted in a proportion of human lung cancers and has been shown to inhibit cell cycle progression as an endogenous cellular regulator of the p53 protein, raising the possibility that it might constitute an additional lung tumor suppressor gene at the 9p21 locus. To test the candidacy of p14ARF as a lung cancer suppressor and assess the role it plays in radiosensitivity, we transfected the wild-type p14ARF gene into four cell lines which had various endogenous gene backgrounds of INK4A-/p53+/RB+ (A549 and H460), INK4A+/p53+/RB- (H446) as well as p14ARF+/p53-/RB+ (Calu-1). We found that transfection of p14ARF is related to an obvious growth inhibition in all wtp53 cell lines, regardless of INK4A/ARF and RB status. Although it has been shown that p53-induced G1 checkpoint in response to DNA damage by ionizing radiation is p14ARF-independent, we found the radiosensitivity of two p14ARF-deficient cell lines was increased after p14ARF gene transfer. The results indicated that cell cycle redistribution after acquiring the exogenous gene might be the main explanation for the enhanced sensitization. An increased radiation-induced apoptotic proportion in one cell line also suggested a fortified p53 function that might be triggered by the restored p14ARF protein.

Amelanotic blue nevus: a variant of blue nevus.

Blue nevi are typically heavily melanized. We report a variant of blue nevus that is minimally pigmented. Of the 1,358 blue nevi seen in our laboratory during the last 6 years, 38 (2.7%) were selected that had scant or absent melanin. We refer to these blue nevi as the amelanotic type. Approximately half of the cases in clinical diagnosis were nevus of some type, whereas other differential diagnoses were basal cell carcinoma, dermatofibroma, and lesion. Histologically all specimens were characterized by the spindle-shaped cells seen in blue nevi, but with very little or no obvious melanin. Some lesions were markedly cellular, resembling the features of cellular blue nevus. No hemosiderin was identified on Perls' stain, whereas Fontana-Masson stain was variably positive. Usually there was fibrous stroma. In most cases, the histologic differential diagnosis was dermatofibroma. Other histologic differential diagnoses included amelanotic and/or spindle cell melanoma, dermal Spitz nevus, neurofibroma, and scar. There was no pleomorphism or increased mitotic activity. Evidence of epidermal melanocytic hyperplasia was seen in two cases. Furthermore, the lesions had been present for many years without evidence of recent change. Immunohistochemistry showed all cases to be strongly positive with anti Mel-5 antibody, but only weakly positive or negative with anti S-100 and HMB-45 antibodies. We would like dermatologists and pathologists to be aware of this unusual and uncommon entity.

Down-regulation of beta1,4-galactosyltransferase gene expression by cell-cycle suppressor gene p16.

Beta1,4-Galactosyltranferase (beta1,4GT, EC 2.4.1.38) is one of the key enzymes controlling the biosynthesis of complex-type oligosaccharides, and is also one of the best-studied glycosyltransferases. To study the molecular mechanisms involved in the regulation of beta1,4GT gene expression, we transfected cell-cycle suppressor gene p16 into A549 cell line (in which p16 is deleted), measured beta1,4GT gene expression by Northern blot hybridization, and evaluated its activity. It was found that p16 could down-regulate beta1,4GT gene expression and its activity. However, p16 decreased cell surface beta1,4GT activity more than total activity. beta1,4GT mRNA stability was also assayed. It was found that p16 could not influence beta1,4GT mRNA stability.

Restoration of the p16 gene is related to increased radiosensitivity of p16-deficient lung adenocarcinoma cell lines.

To assess the role played by p16 gene expression in the radiosensitivity of human lung cancers, we transferred exogenous p16 genes into p16-deficient H460 and A549 lung adenocarcinoma cell lines and compared the cell survival curve in vitro after irradiation. The surviving fraction of the p16-transfected A549p16 and H460p16 cells that expressed exogenous p16 mRNA or protein was lower than those of the parental and negative control cells. The rapid exit of the p16-transfected cells from the G2/M phase in the cell cycle, both before and after irradiation, possibly contributes to the increased radiosensitivity of our experimental p16-transfected lung adenocarcinoma cell lines. We conclude that exogenous p16 gene may be another important factor controlling the intrinsic cellular radiosensitivity of cancer.

Observation of changes in peripheral T-lymphocyte subsets by flow cytometry in patients with liver cancer treated with radioimmunotherapy.

An analysis of lymphocyte subsets was performed in 24 patients with surgically verified unresectable hepatocellular carcinoma (HCC). In all cases, the hepatic artery was cannulated and ligated and a single dose of 131I-Hepama-1 monoclonal antibody (MAb) was infused through a hepatic artery catheter. Blood samples were obtained before infusion and 1 month post-infusion. The results indicated that the CD3+ T-cells (pan T-cells) were normal, whereas the CD4+ T-cells (T-helper/inducer cells) decreased and the CD8+ T-cells (T-suppressor/cytotoxic cells) increased significantly in comparison with the control group. So the CD4+:CD8+ ratio declined markedly. One month post-radioimmunotherapy (RIT), the T-cell subsets did not change, but CD4+, CD8+ and the CD4+:CD8+ ratio differed significantly between patients with and without sequential resection. The changes in the T-cell subsets were closely related to tumour capsule status and the human anti-murine antibody (HAMA) response, but no relationship was found between the tumour and administration of a moderate dose of radioimmunoconjugate. Therefore, determination of changes in the T-lymphocyte subsets and the CD4+:CD8+ ratio could be used as clinical and prognostic indicators in patients who have received RIT. Furthermore, RIT did not affect the distribution of T-cell subsets in patients with HCC.

Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1.

Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.

[Biological characteristics of tumor growth delay in human nasopharyngeal carcinoma xenograft (NPC-837) after irradiation].

Tumor growth and cell cycle of human nasopharyngeal carcinoma xenograft in nude mice (NPC-837) after irradiation were studied. After cobalt-60 irradiation of 5.0Gy, 7.5Gy, 10Gy, 15Gy and 20Gy, the tumor volume was reduced to 80, 70, 50, 35 and 10 percent of the control, respectively. However, the tumors began to grow again. The time (in days) the tumors took to reach the volume of the control was radiation dose-dependent. With 20 Gy irradiation the tumor volume was kept at 10% of the control level for as long as 20 days before regrowth occurred, but it never reached the control level up to 108 days of observation. A mean G2 cell block rate of 1.21% after irradiation was observed and a G2 cell block was marked after 7.5Gy irradiation. The results indicate that NPC-837 is a very radiosensitive and valuable model for the radiobiological research of solid tumors.

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma.

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5'-flanking region.

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.

[Changes in cell cycle of human hepatoma xenograft in nude mice after irradiation].

Cell kinetics in human hepatoma xenograft in nude mice after gamma irradiation was studied using flow cytometry (FCM) method and the changes of AFP in the xenograft were measured by radioimmunological assay. After 10 Gy irradiation, a marked tumor growth delay for 10 days was observed. Cell cycle analysis revealed an acute but temporary block of cell cycling at G2. About 58% cells were in the G2 phase lasting for 90 hours post-irradiation. A concomitant decrease in serum AFP determined by RIA was also observed. The results indicate that the human hepatoma was quite radio-sensitive.

DNA flow cytometric analysis of human nasopharyngeal carcinoma in nude mice.

The tissue from a patient with nasopharyngeal carcinoma has been transferred to nude mice (BALB/c), and has successfully been growing through twenty passages. The tumors in the nude mice and primary human tumor have been examined for the cellular DNA content by FCM and also conventional pathological examination, chromosome analysis, and EBV test. The tumor take rate varied markedly in different passages with a mean value of approximately 70%, and showing a tendency to increase. The tumor doubling time within 6-12 weeks after transplantation of six tumors in 18 and 20 passages were 14.8 and 9.3 days respectively. However, the tumor volume at 12 weeks varied significantly, ranging from 438 to 1998 mm3. By FCM, it has been found that the values of DNA index were about the same in both primary tumor and the tumors in nude mice. The distribution of various phase cells in cell cycle was also about the same in both. In conclusion, the application of FCM to examine the cellular DNA content of the tumor in nude mice is a rapid and sensitive method, useful in the investigation on the stability of biological characteristics of human NPC in nude mice and in further studies on the effects by various therapeutic methods.

[Reactivity of monoclonal antibodies ALT-01 and ALT-04 and identification of lung cancer-associated antigens].

Two IgG1 type monoclonal antibodies ALT-01 and ALT-04 were prepared by two different immunization schedules. ALT-01 was generated by fusing murine myeloma NS-1 cells with splenocytes from a BALB/c mouse immunized by human lung squamous carcinoma cells, which were coated by antisera to mixed human lymphocytes. For preparation of ALT-04, human lung squamous carcinoma xenograft-bearing nude mice were injected I. P. with the spleen cells of normal BALB/c mice in order to acquire immunofunction. The spleen cells from these tumor-bearing nude mice were fused with NS-1 cells. Then, these hybridomas were screened and cloned for 3 times. Two antibodies were shown to recognize the surface antigen on human lung carcinoma cells and several kinds of tumor cell lines but not those on normal cell lines. ALT-01 reacted to neither human lung carcinoma tissue nor its xenograft. ALT-04 reacted to human lung carcinoma tissue, of which, reaction to adenocarcinoma was the strongest but not to various normal tissues. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and autoradiography was used to detect the associated antigen in 35S-labeled human lung carcinoma cells. Antigens, reacting to ALT-01, show one band of Mr 38,000 but those to ALT-04 reveal two bands of Mr 48,000 and 36,000.

[Flow cytometer (FCM) in biological characteristics of nasopharyngeal carcinoma xenografts in nude mice].

Tumor tissue from a patient with nasopharyngeal carcinoma was transplanted to nude mice (BALB/c) and had successfully been maintained through ten passages. The nude mice tumor and the primary human tumor were assayed by FCM for the DNA content in addition to conventional pathological examination, chromosome analysis and EBV assay. The tumor incidence in the different passages showed a marked change ranging from 25% to 81% with a mean value of 52% which tended to increase, however. The doubling time of six tumors in the ninth passage showed no great change 6-12 weeks after transplantation with a mean about 4.3 days. But at week 12, there was a significant difference between each tumor volume, ranging from 438 to 1,998 mm3. By FCM, it was found that the DNA index remained constant in both the primary and nude mice tumors. Among the nude mice tumors in different passages, the distribution of various phase cells in cell cycle was similar to that of the primary tumor. In conclusion, the use of FCM to assay the cellular DNA content in nude mice tumor is rapid and sensitive. It is helpful, for the nude mice tumor, in identifying stability of the biological characteristics and in studying of the mechanism of chemotherapy and radiotherapy in the future.

Isolation and characterization of a rabbit sperm tail protein.

Rabbit sperm tails were obtained by a nitrogen cavitation procedure and separated by discontinuous sucrose gradient centrifugation. Proteins were extracted from sperm tails with 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and separative disk electrophoresis and DEAE-trisacryl column chromatography. A protein with an estimated mol wt of 20.1 +/- 1.1 kD was isolated and found to be homogeneous by SDS-PAGE and designated as rSMP-B. The isoelectric point of rSMP-B was in the range of pH 4.4-4.7. The amino acid composition was determined, and glycine was identified as the N-terminal residue. Antisera were raised against purified rSMP-B. Using a peroxidase-antiperoxidase method, the rSMP-B antigen was located on the surface of the midpiece and tail of the sperm. Testis sections showed intense staining of late spermatids located within the seminiferous tubules. Adult male rabbits were inoculated with rSMP-B protein and Freund's adjuvant. The testis and epididymis of the immunized animals showed absence of sperm. The immunolocalization findings and the immunization data suggest that rSMP-B is formed in late spermatid and that it is an essential structural component of sperm.